Why is DNA replication not 100% accurate

Why is DNA replication not 100% accurate

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I've been reading about DNA mismatch repair (MMR) and how this process improves DNA fidelity. However, I was wondering, what is stopping MMR from correcting all mistakes in the daughter DNA with 100% fidelity? Why is the error rate still around 1 in 10^9 base pairs? Is it because the MMR proteins aren't present in cells in a high enough concentration? What would you have to change about this process to achieve 100% fidelity?

Nothing is 100% precise - any measurement or process allow for some error, the only difference is how often such errors occur (i.e., the probability of an error). These wildely range in biology - e.g., it is about 1 per $10^4$ in HIV replication, but only 1 per $10^9$ for human DNA (due to the repair mechanisms).

Note that $1$ in $10^9$ for human DNA means pretty much that there is about 1 error per each copying of the genome (which has the size of about $3 imes10^9$). One could therefore still pose a question of why the error rate is so high? E.g., Human body consists of about $15 imes10^{12}$ cells. Thus, if the error rate were about 1 per $10^{22}$($approx 3 imes10^9$ by $15 imes10^{12}$), we could have human organisms consisting of the cells with identical DNA.

The answer to this is that errors are not necessarily bad: the copying errors are the source of the mutations driving the evolution! Note also that many of the errors have no effect at all on the well-being of the organism. Thus, it is fair to say that the existing error rates were selected by the evolution for assuring the appropriate rate of the evolutionary change/adaptation, without causing immediate harm to the organism. (One could even speculate that 1 error per genome copy is the appropriate rate across the organisms, but for a moment I cannot support this assertion by references).

Why is DNA replication not 100% accurate - Biology

DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand.

Learning Objectives

Explain how the Meselson and Stahl experiment conclusively established that DNA replication is semi-conservative.

Key Takeaways

Key Points

  • There were three models suggested for DNA replication: conservative, semi-conservative, and dispersive.
  • The conservative method of replication suggests that parental DNA remains together and newly-formed daughter strands are also together.
  • The semi-conservative method of replication suggests that the two parental DNA strands serve as a template for new DNA and after replication, each double-stranded DNA contains one strand from the parental DNA and one new (daughter) strand.
  • The dispersive method of replication suggests that, after replication, the two daughter DNAs have alternating segments of both parental and newly-synthesized DNA interspersed on both strands.
  • Meselson and Stahl, using E. coli DNA made with two nitrogen istopes ( 14 N and 15 N) and density gradient centrifugation, determined that DNA replicated via the semi-conservative method of replication.

Key Terms

  • DNA replication: a biological process occuring in all living organisms that is the basis for biological inheritance
  • isotope: any of two or more forms of an element where the atoms have the same number of protons, but a different number of neutrons within their nuclei

Basics of DNA Replication

Watson and Crick’s discovery that DNA was a two-stranded double helix provided a hint as to how DNA is replicated. During cell division, each DNA molecule has to be perfectly copied to ensure identical DNA molecules to move to each of the two daughter cells. The double-stranded structure of DNA suggested that the two strands might separate during replication with each strand serving as a template from which the new complementary strand for each is copied, generating two double-stranded molecules from one.

Models of Replication

There were three models of replication possible from such a scheme: conservative, semi-conservative, and dispersive. In conservative replication, the two original DNA strands, known as the parental strands, would re-basepair with each other after being used as templates to synthesize new strands and the two newly-synthesized strands, known as the daughter strands, would also basepair with each other one of the two DNA molecules after replication would be “all-old” and the other would be “all-new”. In semi-conservative replication, each of the two parental DNA strands would act as a template for new DNA strands to be synthesized, but after replication, each parental DNA strand would basepair with the complementary newly-synthesized strand just synthesized, and both double-stranded DNAs would include one parental or “old” strand and one daughter or “new” strand. In dispersive replication, after replication both copies of the new DNAs would somehow have alternating segments of parental DNA and newly-synthesized DNA on each of their two strands.

Suggested Models of DNA Replication: The three suggested models of DNA replication. Grey indicates the original parental DNA strands or segments and blue indicates newly-synthesized daughter DNA strands or segments.

To determine which model of replication was accurate, a seminal experiment was performed in 1958 by two researchers: Matthew Meselson and Franklin Stahl.

Meselson and Stahl

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N) that is incorporated into nitrogenous bases and, eventually, into the DNA. The E. coli culture was then shifted into medium containing the common “light” isotope of nitrogen ( 14 N) and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge in a tube in which a cesium chloride density gradient had been established. Some cells were allowed to grow for one more life cycle in 14 N and spun again.

Meselson and Stahl: Meselson and Stahl experimented with E. coli grown first in heavy nitrogen ( 15 N) then in ligher nitrogen ( 14 N.) DNA grown in 15 N (red band) is heavier than DNA grown in 14 N (orange band) and sediments to a lower level in the cesium chloride density gradient in an ultracentrifuge. When DNA grown in 15 N is switched to media containing 14 N, after one round of cell division the DNA sediments halfway between the 15 N and 14 N levels, indicating that it now contains fifty percent 14 N and fifty percent 15 N.. In subsequent cell divisions, an increasing amount of DNA contains 14 N only. These data support the semi-conservative replication model.

During the density gradient ultracentrifugation, the DNA was loaded into a gradient (Meselson and Stahl used a gradient of cesium chloride salt, although other materials such as sucrose can also be used to create a gradient) and spun at high speeds of 50,000 to 60,000 rpm. In the ultracentrifuge tube, the cesium chloride salt created a density gradient, with the cesium chloride solution being more dense the farther down the tube you went. Under these circumstances, during the spin the DNA was pulled down the ultracentrifuge tube by centrifugal force until it arrived at the spot in the salt gradient where the DNA molecules’ density matched that of the surrounding salt solution. At the point, the molecules stopped sedimenting and formed a stable band. By looking at the relative positions of bands of molecules run in the same gradients, you can determine the relative densities of different molecules. The molecules that form the lowest bands have the highest densities.

DNA from cells grown exclusively in 15 N produced a lower band than DNA from cells grown exclusively in 14 N. So DNA grown in 15 N had a higher density, as would be expected of a molecule with a heavier isotope of nitrogen incorporated into its nitrogenous bases. Meselson and Stahl noted that after one generation of growth in 14 N (after cells had been shifted from 15 N), the DNA molecules produced only single band intermediate in position in between DNA of cells grown exclusively in 15 N and DNA of cells grown exclusively in 14 N. This suggested either a semi-conservative or dispersive mode of replication. Conservative replication would have resulted in two bands one representing the parental DNA still with exclusively 15 N in its nitrogenous bases and the other representing the daughter DNA with exclusively 14 N in its nitrogenous bases. The single band actually seen indicated that all the DNA molecules contained equal amounts of both 15 N and 14 N.

The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N and the other corresponded to the band of exclusively 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Dispersive replication would have resulted in exclusively a single band in each new generation, with the band slowly moving up closer to the height of the 14 N DNA band. Therefore, dispersive replication could also be ruled out.

Meselson and Stahl’s results established that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are synthesized. The new strand will be complementary to the parental or “old” strand and the new strand will remain basepaired to the old strand. So each “daughter” DNA actually consists of one “old” DNA strand and one newly-synthesized strand. When two daughter DNA copies are formed, they have the identical sequences to one another and identical sequences to the original parental DNA, and the two daughter DNAs are divided equally into the two daughter cells, producing daughter cells that are genetically identical to one another and genetically identical to the parent cell.

The History of DNA

Figure 1. Friedrich Miescher (1844–1895) discovered nucleic acids.

Modern understandings of DNA have evolved from the discovery of nucleic acid to the development of the double-helix model. In the 1860s, Friedrich Miescher (Figure 1), a physician by profession, was the first person to isolate phosphate-rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.

To see Miescher conduct an experiment step-by-step, click through this review of how he discovered the key role of DNA and proteins in the nucleus.

A half century later, British bacteriologist Frederick Griffith was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally,” rather than by descent. In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing morphology and physiology. He was working with Streptococcus pneumoniae, the bacterium that causes pneumonia. Griffith worked with two strains, rough (R) and smooth (S). The R strain is non-pathogenic (does not cause disease) and is called rough because its outer surface is a cell wall and lacks a capsule as a result, the cell surface appears uneven under the microscope. The S strain is pathogenic (disease-causing) and has a capsule outside its cell wall. As a result, it has a smooth appearance under the microscope. Griffith injected the live R strain into mice and they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle (Figure 2). These experiments are now famously known as Griffith’s transformation experiments.

Figure 2. Two strains of S. pneumoniae were used in Griffith’s transformation experiments. The R strain is non-pathogenic. The S strain is pathogenic and causes death. When Griffith injected a mouse with the heat-killed S strain and a live R strain, the mouse died. The S strain was recovered from the dead mouse. Thus, Griffith concluded that something had passed from the heat-killed S strain to the R strain, transforming the R strain into S strain in the process. (credit “living mouse”: modification of work by NIH credit “dead mouse”: modification of work by Sarah Marriage)

Scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) were interested in exploring this transforming principle further. They isolated the S strain from the dead mice and isolated the proteins and nucleic acids, namely RNA and DNA, as these were possible candidates for the molecule of heredity. They conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.

Forensic Scientists and DNA Analysis

DNA evidence was used for the first time to solve an immigration case. The story started with a teenage boy returning to London from Ghana to be with his mother. Immigration authorities at the airport were suspicious of him, thinking that he was traveling on a forged passport. After much persuasion, he was allowed to go live with his mother, but the immigration authorities did not drop the case against him. All types of evidence, including photographs, were provided to the authorities, but deportation proceedings were started nevertheless. Around the same time, Dr. Alec Jeffreys of Leicester University in the United Kingdom had invented a technique known as DNA fingerprinting. The immigration authorities approached Dr. Jeffreys for help. He took DNA samples from the mother and three of her children, plus an unrelated mother, and compared the samples with the boy’s DNA. Because the biological father was not in the picture, DNA from the three children was compared with the boy’s DNA. He found a match in the boy’s DNA for both the mother and his three siblings. He concluded that the boy was indeed the mother’s son.

Forensic scientists analyze many items, including documents, handwriting, firearms, and biological samples. They analyze the DNA content of hair, semen, saliva, and blood, and compare it with a database of DNA profiles of known criminals. Analysis includes DNA isolation, sequencing, and sequence analysis most forensic DNA analysis involves polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci and electrophoresis to determine the length of the PCR-amplified fragment. Only mitochondrial DNA is sequenced for forensics. Forensic scientists are expected to appear at court hearings to present their findings. They are usually employed in crime labs of city and state government agencies. Geneticists experimenting with DNA techniques also work for scientific and research organizations, pharmaceutical industries, and college and university labs. Students wishing to pursue a career as a forensic scientist should have at least a bachelor’s degree in chemistry, biology, or physics, and preferably some experience working in a laboratory.

Experiments conducted by Martha Chase and Alfred Hershey in 1952 provided confirmatory evidence that DNA was the genetic material and not proteins. Chase and Hershey were studying a bacteriophage, which is a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA. The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages. Hershey and Chase labeled one batch of phage with radioactive sulfur, 35 S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32 P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus.

Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to be detached from the host cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the tube that contained phage labeled with 35 S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32 P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins (Figure 3).

Figure 3. In Hershey and Chase’s experiments, bacteria were infected with phage radiolabeled with either 35 S, which labels protein, or 32 P, which labels DNA. Only 32 P entered the bacterial cells, indicating that DNA is the genetic material.

Around this same time, Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. This is also known as Chargaff’s rules. This finding proved immensely useful when Watson and Crick were getting ready to propose their DNA double helix model.

Practice Question

The experiments by Hershey and Chase helped confirm that DNA was the hereditary material on the basis of the finding that:

  1. radioactive phage were found in the pellet
  2. radioactive cells were found in the supernatant
  3. radioactive sulfur was found inside the cell
  4. radioactive phosphorus was found in the cell

What would happen if DNA replication occurred incorrectly?

All this is further explained here. Accordingly, what errors can occur in DNA replication?

These types of errors include depurination, which occurs when the bond connecting a purine to its deoxyribose sugar is broken by a molecule of water, resulting in a purine-free nucleotide that can't act as a template during DNA replication, and deamination, which results in the loss of an amino group from a nucleotide,

Beside above, why is it important for DNA replication to occur without any mistakes? -To prevent mutations and keeping organisms in a healthy state. Explanation: DNA replication involves synthesis of daughter DNA molecules by using parent DNA as template. The cells have many methods to proceed error free replication, thus, DNA replication shows a highly accurate process.

Also to know is, why is it important for the cell to correct any errors that occur during replication?

Mutations during replication and damage after replication make it necessary for there to be a repair system to fix any errors in newly synthesized DNA. Radiation damage which can lead to nicks in the backbone of DNA or the formation of thymine dimers, which will be discussed later.

What happens when DNA is damaged?

The DNA in just one of your cells gets damaged tens of thousands of times per day. Because DNA provides the blueprint for the proteins your cells need to function, this damage can cause serious issues&mdashincluding cancer. Fortunately, your cells have ways of fixing most of these problems, most of the time.

The Immortal Strand

When a cell divides, it replicates its DNA by splitting apart double-stranded DNA and makes new copies along the single strands of the original DNA. The original DNA, which is now part of a double-strand containing new DNA, randomly splits between the two dividing cells. Some types of adult stem cells, however, have non-random segregation of the original DNA strand. By always keeping the original DNA strands, it ensures that it maintains the original information. The original DNA strands are referred to as the immortal strands.


Three hypotheses had been previously proposed for the method of replication of DNA.

In the semiconservative hypothesis, proposed by Watson and Crick, the two strands of a DNA molecule separate during replication. Each strand then acts as a template for synthesis of a new strand. [2]

The conservative hypothesis proposed that the entire DNA molecule acted as a template for the synthesis of an entirely new one. According to this model, histone proteins bind to the DNA, revolving the strand and exposing the nucleotide bases (which normally line the interior) for hydrogen bonding. [3]

The dispersive hypothesis is exemplified by a model proposed by Max Delbrück, which attempts to solve the problem of unwinding the two strands of the double helix by a mechanism that breaks the DNA backbone every 10 nucleotides or so, untwists the molecule, and attaches the old strand to the end of the newly synthesized one. This would synthesize the DNA in short pieces alternating from one strand to the other. [4]

Each of these three models makes a different prediction about the distribution of the "old" DNA in molecules formed after replication. In the conservative hypothesis, after replication, one molecule is the entirely conserved "old" molecule, and the other is all newly synthesized DNA. The semiconservative hypothesis predicts that each molecule after replication will contain one old and one new strand. The dispersive model predicts that each strand of each new molecule will contain a mixture of old and new DNA. [5]

Nitrogen is a major constituent of DNA. 14 N is by far the most abundant isotope of nitrogen, but DNA with the heavier (but non-radioactive) 15 N isotope is also functional.

E. coli was grown for several generations in a medium containing NH4Cl with 15 N. When DNA is extracted from these cells and centrifuged on a salt (CsCl) density gradient, the DNA separates out at the point at which its density equals that of the salt solution. The DNA of the cells grown in 15 N medium had a higher density than cells grown in normal 14 N medium. After that, E. coli cells with only 15 N in their DNA were transferred to a 14 N medium and were allowed to divide the progress of cell division was monitored by microscopic cell counts and by colony assay.

DNA was extracted periodically and was compared to pure 14 N DNA and 15 N DNA. After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. However, this result was consistent with both semiconservative and dispersive replication. Semiconservative replication would result in double-stranded DNA with one strand of 15 N DNA, and one of 14 N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of 15 N and 14 N DNA, either of which would have appeared as DNA of an intermediate density.

The authors continued to sample cells as replication continued. DNA from cells after two replications had been completed was found to consist of equal amounts of DNA with two different densities, one corresponding to the intermediate density of DNA of cells grown for only one division in 14 N medium, the other corresponding to DNA from cells grown exclusively in 14 N medium. This was inconsistent with dispersive replication, which would have resulted in a single density, lower than the intermediate density of the one-generation cells, but still higher than cells grown only in 14 N DNA medium, as the original 15 N DNA would have been split evenly among all DNA strands. The result was consistent with the semiconservative replication hypothesis. [6]

7.2: Semi-Conservative DNA Replication

  • Contributed by E. V. Wong
  • Axolotl Academica Publishing (Biology) at Axolotl Academica Publishing

DNA replication is similar to transcription in its most general idea: a polymerase enzyme reads a strand of DNA one nucleotide at a time, it takes a random nucleotide from the nucleoplasm, and if it is complementary to the nucleotide in the DNA, the polymerase adds it to the new strand it is creating. Of course, there are significant differences between replication and transcription too, not the least of which is that both strands of DNA are being read simultaneously in order to create two new complementary strands that will eventually result in a complete and nearly perfect copy of an entire organismal genome.

Figure (PageIndex<7>). DNA replication. Prior to the discovery of the enzymes involved in replication, three general mechanisms were proposed. In conservative replication, the original DNA strands stay associated with each other, while the newly made DNA forms its own double-helix. Semi-conservative replication posits the creation of hybrid old-new double helices. Dispersive replication proposed molecules composed of randomized fragments of double-old and double-new DNA.

One of the most important concepts of DNA replication is that it is a semi-conservative process (Figure (PageIndex<7>)). This means that every double helix in the new generation of an organism consists of one complete &ldquoold&rdquo strand and one complete &ldquonew&rdquo strand wrapped around each other. This is in contrast to the two other possible models of DNA replication, the conservative model, and the dispersive model. A conservative mechanism of replication proposes that the old DNA is used as a template only and is not incorporated into the new double-helix. Thus the new cell has one completely new double-helix and one completely old double-helix. The dispersive model of replication posits a final product in which each double helix of DNA is a mixture of fragments of old and new DNA. In light of current knowledge, it is difficult to imagine a dispersive mechanism, but at the time, there were no mechanistic models at all. The Meselson-Stahl experiments (1958) clearly demonstrated that the mechanism must be semi-conservative, and this was confirmed once the key enzymes were discovered and their mechanisms elucidated.

In the Meselson-Stahl experiments, E. coli were first incubated with 15 N, a heavy isotope of nitrogen. Although it is only a difference in mass of one neutron per atom, there is a great enough difference in mass between heavy nitrogen-containing DNA (in the purine and pyrimidine bases) and light/normal nitrogen-containing DNA that they can be separated from one another by ultracentrifugation through a CsCl concentration gradient (Figure (PageIndex<7>)).

Over 14 generations, this led to a population of E. coli that had heavy nitrogen incorporated into all of the DNA (shown in blue). Then, the bacteria are grown for one or two divisions in &ldquolight&rdquo nitrogen, 14 N. When the DNA from the bacterial populations was examined by centrifugation, it was found that instead of light DNA and heavy DNA, as would be expected if DNA replications was conservative, there was a single band in and intermediate position on the gradient. This supports a semi-conservative model in which each strand of original DNA not only acts as a template for making new DNA, it is itself incorporated into the new double-helix.


Damage to DNA that occurs naturally can result from metabolic or hydrolytic processes. Metabolism releases compounds that damage DNA including reactive oxygen species, reactive nitrogen species, reactive carbonyl species, lipid peroxidation products and alkylating agents, among others, while hydrolysis cleaves chemical bonds in DNA. [8] Naturally occurring oxidative DNA damages arise at least 10,000 times per cell per day in humans and as much as 100,000 per cell per day in rats [9] as documented below.

Oxidative DNA damage can produce more than 20 types of altered bases [10] [11] as well as single strand breaks. [12]

Other types of endogeneous DNA damages, given below with their frequencies of occurrence, include depurinations, depyrimidinations, double-strand breaks, O6-methylguanines and cytosine deamination.

DNA can be damaged via environmental factors as well. Environmental agents such as UV light, ionizing radiation, and genotoxic chemicals. Replication forks can be stalled due to damaged DNA and double strand breaks are also a form of DNA damage. [13]

Frequencies Edit

The list below shows some frequencies with which new naturally occurring DNA damages arise per day, due to endogenous cellular processes.

  • Oxidative damages
    • Humans, per cell per day
      • 10,000 [9]
        11,500 [14]
        2,800 [15] specific damages 8-oxoGua, 8-oxodG plus 5-HMUra
        2,800 [16] specific damages 8-oxoGua, 8-oxodG plus 5-HMUra
      • 74,000 [14]
        86,000 [17]
        100,000 [9]
      • 34,000 [15] specific damages 8-oxoGua, 8-oxodG plus 5-HMUra
        47,000 [18] specific damages oxo8dG in mouse liver
        28,000 [16] specific damages 8-oxoGua, 8-oxodG, 5-HMUra
      • Mammalian cells, per cell per day
        • 2,000 to 10,000 [19][20]
          9,000 [21]
          12,000 [22]
          13,920 [23]
        • Mammalian cells, per cell per day
          • 600 [22]
            696 [23]
          • Mammalian cells, per cell per day
            • 55,200 [23]
            • Human cells, per cell cycle
              • 10 [24]
                50 [25]
              • Mammalian cells, per cell per day
                • 3,120 [23]
                • Mammalian cells, per cell per day
                  • 192 [23]

                  Another important endogenous DNA damage is M1dG, short for (3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one). The excretion in urine (likely reflecting rate of occurrence) of M1dG may be as much as 1,000-fold lower than that of 8-oxodG. [26] However, a more important measure may be the steady-state level in DNA, reflecting both rate of occurrence and rate of DNA repair. The steady-state level of M1dG is higher than that of 8-oxodG. [27] This points out that some DNA damages produced at a low rate may be difficult to repair and remain in DNA at a high steady-state level. Both M1dG [28] and 8-oxodG [29] are mutagenic.

                  Steady-state levels Edit

                  Steady-state levels of DNA damages represent the balance between formation and repair. More than 100 types of oxidative DNA damage have been characterized, and 8-oxodG constitutes about 5% of the steady state oxidative damages in DNA. [18] Helbock et al. [14] estimated that there were 24,000 steady state oxidative DNA adducts per cell in young rats and 66,000 adducts per cell in old rats. This reflects the accumulation of DNA damage with age. DNA damage accumulation with age is further described in DNA damage theory of aging.

                  Swenberg et al. [30] measured average amounts of selected steady state endogenous DNA damages in mammalian cells. The seven most common damages they evaluated are shown in Table 1.

                  Table 1. Steady-state amounts of endogenous DNA damages
                  Endogenous lesions Number per cell
                  Abasic sites 30,000
                  N7-(2-hydroxethyl)guanine (7HEG) 3,000
                  8-hydroxyguanine 2,400
                  7-(2-oxoethyl)guanine 1,500
                  Formaldehyde adducts 960
                  Acrolein-deoxyguanine 120
                  Malondialdehyde-deoxyguanine 60

                  Evaluating steady-state damages in specific tissues of the rat, Nakamura and Swenberg [31] indicated that the number of abasic sites varied from about 50,000 per cell in liver, kidney and lung to about 200,000 per cell in the brain.

                  Proteins promoting endogenous DNA damage were identified in a 2019 paper as the DNA "damage-up" proteins (DDPs). [32] The DDP mechanisms fall into 3 clusters:

                  • reactive oxygen increase by transmembrane transporters,
                  • chromosome loss by replisome binding,
                  • replication stalling by transcription factors. [32]

                  The DDP human homologs are over-represented in known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. [32]

                  In the presence of DNA damage, the cell can either repair the damage or induce cell death if the damage is beyond repair.

                  Types Edit

                  The seven main types of DNA repair and one pathway of damage tolerance, the lesions they address, and the accuracy of the repair (or tolerance) are shown in this table. For a brief description of the steps in repair see DNA repair mechanisms or see each individual pathway.

                  Major pathways of DNA repair and one tolerance mechanism
                  Repair pathway Lesions Accuracy Ref.
                  Base excision repair corrects DNA damage from oxidation, deamination and alkylation, also single-strand breaks accurate [33] [34]
                  Nucleotide excision repair oxidative endogenous lesions such as cyclopurine, sunlight-induced thymine dimers (cyclobutane dimers and pyrimidine (6-4) pyrimidone photoproducts) accurate [35] [36] [37]
                  Homology-directed repair double-strand breaks in the mid-S phase or mid-G2 phase of the cell cycle accurate [38]
                  Non-homologous end joining double-strand breaks if cells are in the G0 phase. the G1 phase or the G2 phase of the cell cycle somewhat inaccurate [38]
                  Microhomology-mediated end joining or alt-End joining double-strand breaks in the S phase of the cell cycle always inaccurate [38]
                  DNA mismatch repair base substitution mismatches and insertion-deletion mismatches generated during DNA replication accurate [39]
                  Direct reversal (MGMT and AlkB) 6-O-methylguanine is reversed to guanine by MGMT, some other methylated bases are demethylated by AlkB accurate [40]
                  Translesion synthesis DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions may be inaccurate [41]

                  The schematic diagram indicates the roles of insufficient DNA repair in aging and cancer, and the role of apoptosis in cancer prevention. An excess of naturally occurring DNA damage, due to inherited deficiencies in particular DNA repair enzymes, can cause premature aging or increased risk for cancer (see DNA repair-deficiency disorder). On the other hand, the ability to trigger apoptosis in the presence of excess un-repaired DNA damage is critical for prevention of cancer. [42]

                  DNA repair proteins are often activated or induced when DNA has sustained damage. However, excessive DNA damage can initiate apoptosis (i.e., programmed cell death) if the level of DNA damage exceeds the repair capacity. Apoptosis can prevent cells with excess DNA damage from undergoing mutagenesis and progression to cancer. [43]

                  Inflammation is often caused by infection, such as with hepatitis B virus (HBV), hepatitis C virus (HCV) or Helicobacter pylori. Chronic inflammation is also a central characteristic of obesity. [44] [45] [46] [47] Such inflammation causes oxidative DNA damage. This is due to the induction of reactive oxygen species (ROS) by various intracellular inflammatory mediators. [48] [49] [50] HBV and HCV infections, in particular, cause 10,000-fold and 100,000-fold increases in intracellular ROS production, respectively. [51] Inflammation-induced ROS that cause DNA damage can trigger apoptosis, [52] [53] but may also cause cancer if repair and apoptotic processes are insufficiently protective. [45]

                  Bile acids, stored in the gall bladder, are released into the small intestine in response to fat in the diet. Higher levels of fat cause greater release. [54] Bile acids cause DNA damage, including oxidative DNA damage, double-strand DNA breaks, aneuploidy and chromosome breakage. [55] High-normal levels of the bile acid deoxycholic acid cause apoptosis in human colon cells, [56] but may also lead to colon cancer if repair and apoptotic defenses are insufficient. [57]

                  Apoptosis serves as a safeguard mechanism against tumorigenesis. [58] It prevents the increased mutagenesis that excess DNA damage could cause, upon replication. [59]

                  At least 17 DNA repair proteins, distributed among five DNA repair pathways, have a "dual role" in response to DNA damage. With moderate levels of DNA damage, these proteins initiate or contribute to DNA repair. However, when excessive levels of DNA damage are present, they trigger apoptosis. [43]

                  The packaging of eukaryotic DNA into chromatin is a barrier to all DNA-based processes that require enzyme action. For most DNA repair processes, the chromatin must be remodeled. In eukaryotes, ATP-dependent chromatin remodeling complexes and histone-modifying enzymes are two factors that act to accomplish this remodeling process after DNA damage occurs. [60] Further DNA repair steps, involving multiple enzymes, usually follow. Some of the first responses to DNA damage, with their timing, are described below. More complete descriptions of the DNA repair pathways are presented in articles describing each pathway. At least 169 enzymes are involved in DNA repair pathways. [61]

                  Base excision repair Edit

                  Oxidized bases in DNA are produced in cells treated with Hoechst dye followed by micro-irradiation with 405 nm light. [62] Such oxidized bases can be repaired by base excision repair.

                  When the 405 nm light is focused along a narrow line within the nucleus of a cell, about 2.5 seconds after irradiation, the chromatin remodeling enzyme Alc1 achieves half-maximum recruitment onto the irradiated micro-line. [63] The line of chromatin that was irradiated then relaxes, expanding side-to-side over the next 60 seconds. [63]

                  Within 6 seconds of the irradiation with 405 nm light, there is half-maximum recruitment of OGG1 to the irradiated line. [62] OGG1 is an enzyme that removes the oxidative DNA damage 8-oxo-dG from DNA. Removal of 8-oxo-dG, during base excision repair, occurs with a half-life of 11 minutes. [18]

                  Nucleotide excision repair Edit

                  Ultraviolet (UV) light induces the formation of DNA damages including pyrimidine dimers (such as thymine dimers) and 6,4 photoproducts. These types of "bulky" damages are repaired by nucleotide excision repair.

                  After irradiation with UV light, DDB2, in a complex with DDB1, the ubiquitin ligase protein CUL4A and the RING finger protein ROC1, associates with sites of damage within chromatin. Half-maximum association occurs in 40 seconds. [64] PARP1 also associates within this period. [65] The PARP1 protein attaches to both DDB1 and DDB2 and then PARylates (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling protein ALC1. [65] ALC1 relaxes chromatin at sites of UV damage to DNA. In addition, the ubiquitin E3 ligase complex DDB1-CUL4A carries out ubiquitination of the core histones H2A, H3, and H4, as well as the repair protein XPC, which has been attracted to the site of the DNA damage. [66] XPC, upon ubiquitination, is activated and initiates the nucleotide excision repair pathway. Somewhat later, at 30 minutes after UV damage, the INO80 chromatin remodeling complex is recruited to the site of the DNA damage, and this coincides with the binding of further nucleotide excision repair proteins, including ERCC1. [67]

                  Homologous recombinational repair Edit

                  Double-strand breaks (DSBs) at specific sites can be induced by transfecting cells with a plasmid encoding I-SceI endonuclease (a homing endonuclease). Multiple DSBs can be induced by irradiating sensitized cells (labeled with 5'-bromo-2'-deoxyuridine and with Hoechst dye) with 780 nm light. These DSBs can be repaired by the accurate homologous recombinational repair or by the less accurate non-homologous end joining repair pathway. Here we describe the early steps in homologous recombinational repair (HRR).

                  After treating cells to introduce DSBs, the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10. [68] This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites with half-maximum recruitment in well under a second. [68] SIRT6 at the site is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to a DNA break site and for efficient repair of DSBs. [68] PARP1 protein starts to appear at DSBs in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs. [69] This then allows half maximum recruitment of the DNA repair enzymes MRE11 within 13 seconds and NBS1 within 28 seconds. [69] MRE11 and NBS1 carry out early steps of the HRR pathway.

                  γH2AX, the phosphorylated form of H2AX is also involved in early steps of DSB repair. The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin. [70] γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute. [70] The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break. [70] γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation, RNF8 protein can be detected in association with γH2AX. [71] RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4, [72] a component of the nucleosome remodeling and deacetylase complex NuRD.

                  Pause for DNA repair Edit

                  After rapid chromatin remodeling, cell cycle checkpoints may be activated to allow DNA repair to be completed before the cell cycle progresses. First, two kinases, ATM and ATR, are activated within 5 or 6 minutes after DNA is damaged. This is followed by phosphorylation of the cell cycle checkpoint protein Chk1, initiating its function, about 10 minutes after DNA is damaged. [73]

                  The DNA damage 8-oxo-dG does not occur randomly in the genome. In mouse embryonic fibroblasts, a 2 to 5-fold enrichment of 8-oxo-dG was found in genetic control regions, including promoters, 5'-untranslated regions and 3'-untranslated regions compared to 8-oxo-dG levels found in gene bodies and in intergenic regions. [74] In rat pulmonary artery endothelial cells, when 22,414 protein-coding genes were examined for locations of 8-oxo-dG, the majority of 8-oxo-dGs (when present) were found in promoter regions rather than within gene bodies. [75] Among hundreds of genes whose expression levels were affected by hypoxia, those with newly acquired promoter 8-oxo-dGs were upregulated, and those genes whose promoters lost 8-oxo-dGs were almost all downregulated. [75]

                  As reviewed by Wang et al., [76] oxidized guanine appears to have multiple regulatory roles in gene expression. In particular, when oxidative stress produces 8-oxo-dG in the promoter of a gene, the oxidative stress may also inactivate OGG1, an enzyme that targets 8-oxo-dG and normally initiates repair of 8-oxo-dG damage. The inactive OGG1, which no longer excises 8-oxo-dG, nevertheless targets and complexes with 8-oxo-dG, and causes a sharp (

                  70 o ) bend in the DNA. This allows the assembly of a transcriptional initiation complex, up-regulating transcription of the associated gene. [76] [77]

                  When 8-oxo-dG is formed in a guanine rich, potential G-quadruplex-forming sequence (PQS) in the coding strand of a promoter, active OGG1 excises the 8-oxo-dG and generates an apurinic/apyrimidinic site (AP site). The AP site enables melting of the duplex to unmask the PQS, adopting a G-quadruplex fold (G4 structure/motif) that has a regulatory role in transcription activation. [76] [78]

                  When 8-oxo-dG is complexed with active OGG1 it may then recruit chromatin remodelers to modulate gene expression. Chromodomain helicase DNA-binding protein 4 (CHD4), a component of the (NuRD) complex, is recruited by OGG1 to oxidative DNA damage sites. CHD4 then attracts DNA and histone methylating enzymes that repress transcription of associated genes. [76]

                  Oxidation of guanine Edit

                  Oxidation of guanine, particularly within CpG sites, may be especially important in learning and memory. Methylation of cytosines occurs at 60–90% of CpG sites depending on the tissue type. [79] In the mammalian brain,

                  62% of CpGs are methylated. [79] Methylation of CpG sites tends to stably silence genes. [80] More than 500 of these CpG sites are de-methylated in neuron DNA during memory formation and memory consolidation in the hippocampus [81] [82] and cingulate cortex [82] regions of the brain. As indicated below, the first step in de-methylation of methylated cytosine at a CpG site is oxidation of the guanine to form 8-oxo-dG.

                  Role of oxidized guanine in DNA de-methylation Edit

                  The figure in this section shows a CpG site where the cytosine is methylated to form 5-methylcytosine (5mC) and the guanine is oxidized to form 8-oxo-2'-deoxyguanosine (in the figure this is shown in the tautomeric form 8-OHdG). When this structure is formed, the base excision repair enzyme OGG1 targets 8-OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp-8-OHdG site recruits TET1, and TET1 oxidizes the 5mC adjacent to the 8-OHdG. This initiates de-methylation of 5mC. [83] TET1 is a key enzyme involved in de-methylating 5mCpG. However, TET1 is only able to act on 5mCpG if the guanine was first oxidized to form 8-hydroxy-2'-deoxyguanosine (8-OHdG or its tautomer 8-oxo-dG), resulting in a 5mCp-8-OHdG dinucleotide (see figure in this section). [83] This initiates the de-methylation pathway on the methylated cytosine, finally resulting in an unmethylated cytosine (see DNA oxidation for further steps in forming unmethylated cytosine).

                  Altered protein expression in neurons, due to changes in methylation of DNA, (likely controlled by 8-oxo-dG-dependent de-methylation of CpG sites in gene promoters within neuron DNA) has been established as central to memory formation. [84]

                  Role of double-strand breaks in memory formation Edit

                  Generation of Neuronal Activity-Related DSBs

                  Double-stranded breaks (DSBs) in regions of DNA related to neuronal activity are produced by a variety of mechanisms within and around the genome. The enzyme topoisomerase II, or TOPIIβ plays a key role in DSB formation by aiding in the demethylation or loosening of histones wrapped around the double helix to promote transcription. [85] Once the chromatin structure is opened, DSBs are more likely to accumulate, however, this is normally repaired by TOPIIβ through its intrinsic religation ability that rejoins the cleaved DNA ends. [85]

                  Failure of TOPIIβ to religase can have drastic consequences on protein synthesis, where it is estimated that “blocking TOPIIβ activity alters the expression of nearly one-third of all developmentally regulated genes,” such as neural immediate early genes (IEGs) involved in memory consolidation. [85] [86] Rapid expression of egr-1, c-Fos, and Arc IEGs have been observed in response to increased neuronal activity in the hippocampus region of the brain where memory processing takes place. [87] As a preventative measure against TOPIIβ failure, DSB repair molecules are recruited via two different pathways: non-homologous end joining (NHEJ) pathway factors, which perform a similar religation function to that of TOPIIβ, and the homologous recombination (HR) pathway, which uses the non-broken sister strand as a template to repair the damaged strand of DNA. [85] [88]

                  Stimulation of neuronal activity, as previously mentioned in IEG expression, is another mechanism through which DSBs are generated. Changes in level of activity have been used in studies as a biomarker to trace the overlap between DSBs and increased histone H3K4 methylation in promoter regions of IEGs. [85] [88] Other studies have indicated that transposable elements (TEs) can cause DSBs through endogenous activity that involves using endonuclease enzymes to insert and cleave target DNA at random sites. [89] [90]

                  DSBs and Memory Reconsolidation

                  While accumulation of DSBs generally inhibits long term memory consolidation, the process of reconsolidation, in contrast, is DSB-dependent. Memory reconsolidation involves the modification of existing memories stored in long-term memory. [91] Research involving NPAS4, a gene that regulates neuroplasticity in the hippocampus during contextual learning and memory formation, has revealed a link between deletions in the coding region and impairments in recall of fear memories in transgenic rats. [85] Moreover, the enzyme H3K4me3, which catalyzes the demethylation of the H3K4 histone, was upregulated at the promoter region of the NPAS4 gene during the reconsolidation process, while knockdown (gene knockdown) of the same enzyme impeded reconsolidation. [85] A similar effect was observed in TOPIIβ, where knockdown also impaired the fear memory response in rats, indicating that DSBs, along with the enzymes that regulate them, influence memory formation at multiple stages.

                  DSBs and Neurodegeneration

                  Buildup of DSBs more broadly leads to the degeneration of neurons, hindering the function of memory and learning processes. Due to their lack of cell division and high metabolic activity, neurons are especially prone to DNA damage. [88] Additionally, an imbalance of DSBs and DNA repair molecules for neuronal-activity genes has been linked to the development of various human neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). [88] In patients with Alzheimer’s disease, DSBs accumulate in neurons at early stages and are the driving force behind memory loss, a key characteristic of the disease. [88] Other external factors that result in increased levels of activity-dependent DSBs in people with AD are oxidative damage to neurons, which can result in more DSBs when multiple lesions occur close to one another. Environmental factors such as viruses and a high-fat diet have also been associated with disrupted function of DNA repair molecules.

                  One targeted therapy for treating patients with AD has involved suppression of the BRCA1 gene in human brains, initially tested in transgenic mice, where DSB levels were observed to have increased and memory loss had occurred, suggesting that BRCA1 could “serve as a therapeutic target for AD and AD-related dementia.” [88] Similarly, the protein ATM involved in DNA repair and epigenetic modifications to the genome is positively correlated with neuronal loss in AD brains, indicating the protein is another key component in the intrinsically-linked processes of neurodegeneration, DSB production, and memory formation. [88]

                  Most damage can be repaired without triggering the damage response system, however more complex damage activates ATR and ATM, key protein kinases in the damage response system. [92] DNA damage inhibits M-CDKs which are a key component of progression into Mitosis.

                  In all eukaryotic cells, ATR and ATM are protein kinases that detect DNA damage. They bind to DNA damaged sites and activate Chk1, Chk2, and, in animal cells, p53. Together, these proteins make up the DNA damage response system. Some DNA damage does not require the recruitment of ATR and ATM, it is only difficult and extensive damage that requires ATR and ATM. ATM and ATR are required for NHEJ, HR, ICL repair, and NER, as well as replication fork stability during unperturbed DNA replication and in response to replication blocks. [7]

                  ATR is recruited for different forms of damage such as nucleotide damage, stalled replication forks and double strand breaks. ATM is specifically for the damage response to double strand breaks. The MRN complex (composed of Mre11, Rad50, and Nbs1) form immediately at the site of double strand break. This MRN complex recruits ATM to the site of damage. ATR and ATM phosphorylate various proteins that contribute to the damage repair system. The binding of ATR and ATM to damage sites on DNA lead to the recruitment of Chk1 and Chk2. These protein kinases send damage signals to the cell cycle control system to delay the progression of the cell cycle. [13]

                  Chk1 leads to the production of DNA repair enzymes. Chk2 leads to reversible cell cycle arrest. Chk2, as well as ATR/ATM, can activate p53, which leads to permanent cell cycle arrest or apoptosis.

                  When there is too much damage, apoptosis is triggered in order to protect the organism from potentially harmful cells.7 p53, also known as a tumor suppressor gene, is a major regulatory protein in the DNA damage response system which binds directly to the promoters of its target genes. p53 acts primarily at the G1 checkpoint (controlling the G1 to S transition), where it blocks cell cycle progression. [6] Activation of p53 can trigger cell death or permanent cell cycle arrest. p53 can also activate certain repair pathways such was NER. [92]

                  Regulation of p53 Edit

                  In the absence of DNA damage, p53 is regulated by Mdm2 and constantly degraded. When there is DNA damage, Mdm2 is phosphorylated, most likely caused by ATM. The phosphorylation of Mdm2 leads to a reduction in the activity of Mdm2, thus preventing the degradation of p53. Normal, undamaged cell, usually has low levels of p53 while cells under stress and DNA damage, will have high levels of p53. [13]

                  P53 serves as transcription factor for bax and p21 Edit

                  p53 serves as a transcription factors for both bax, a proapoptotic protein as well as p21, a CDK inhibitor. CDK Inhibitors result in cell cycle arrest. Arresting the cell provides the cell time to repair the damage, and if the damage is irreparable, p53 recruits bax to trigger apoptosis. [92]

                  DDR and p53 role in cancer Edit

                  p53 is a major key player in the growth of cancerous cells. Damaged DNA cells with mutated p53 are at a higher risk of becoming cancerous. Common chemotherapy treatments are genotoxic. These treatments are ineffective in cancer tumor that have mutated p53 since they do not have a functioning p53 to either arrest or kill the damaged cell.

                  One indication that DNA damages are a major problem for life is that DNA repair processes, to cope with DNA damages, have been found in all cellular organisms in which DNA repair has been investigated. For example, in bacteria, a regulatory network aimed at repairing DNA damages (called the SOS response in Escherichia coli) has been found in many bacterial species. E. coli RecA, a key enzyme in the SOS response pathway, is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. [93] Genes homologous to RecA and to other central genes in the SOS response pathway are found in almost all the bacterial genomes sequenced to date, covering a large number of phyla, suggesting both an ancient origin and a widespread occurrence of recombinational repair of DNA damage. [94] Eukaryotic recombinases that are homologues of RecA are also widespread in eukaryotic organisms. For example, in fission yeast and humans, RecA homologues promote duplex-duplex DNA-strand exchange needed for repair of many types of DNA lesions. [95] [96]

                  Another indication that DNA damages are a major problem for life is that cells make large investments in DNA repair processes. As pointed out by Hoeijmakers, [3] repairing just one double-strand break could require more than 10,000 ATP molecules, as used in signaling the presence of the damage, the generation of repair foci, and the formation (in humans) of the RAD51 nucleofilament (an intermediate in homologous recombinational repair). (RAD51 is a homologue of bacterial RecA.) If the structural modification occurs during the G1 phase of DNA replication, the G1-S checkpoint arrests or postpones the furtherance of the cell cycle before the product enters the S phase. [1]

                  Differentiated somatic cells of adult mammals generally replicate infrequently or not at all. Such cells, including, for example, brain neurons and muscle myocytes, have little or no cell turnover. Non-replicating cells do not generally generate mutations due to DNA damage-induced errors of replication. These non-replicating cells do not commonly give rise to cancer, but they do accumulate DNA damages with time that likely contribute to aging ( see DNA damage theory of aging ). In a non-replicating cell, a single-strand break or other type of damage in the transcribed strand of DNA can block RNA polymerase II-catalysed transcription. [97] This would interfere with the synthesis of the protein coded for by the gene in which the blockage occurred.

                  Brasnjevic et al. [98] summarized the evidence showing that single-strand breaks accumulate with age in the brain (though accumulation differed in different regions of the brain) and that single-strand breaks are the most frequent steady-state DNA damages in the brain. As discussed above, these accumulated single-strand breaks would be expected to block transcription of genes. Consistent with this, as reviewed by Hetman et al., [99] 182 genes were identified and shown to have reduced transcription in the brains of individuals older than 72 years, compared to transcription in the brains of those less than 43 years old. When 40 particular proteins were evaluated in a muscle of rats, the majority of the proteins showed significant decreases during aging from 18 months (mature rat) to 30 months (aged rat) of age. [100]

                  Another type of DNA damage, the double-strand break, was shown to cause cell death (loss of cells) through apoptosis. [101] This type of DNA damage would not accumulate with age, since once a cell was lost through apoptosis, its double-strand damage would be lost with it. Thus, damaged DNA segments undermine the DNA replication machinery because these altered sequences of DNA cannot be utilized as true templates to produce copies of one's genetic material. [1]

                  When DNA is damaged, the cell responds in various ways to fix the damage and minimize the effects on the cell. One such response, specifically in eukaryotic cells, is to delay cell division—the cell becomes arrested for some time in the G2 phase before progressing through the rest of the cell cycle. Various studies have been conducted to elucidate the purpose of this G2 arrest that is induced by DNA damage. Researchers have found that cells that are prematurely forced out of the delay have lower cell viability and higher rates of damaged chromosomes compared with cells that are able to undergo a full G2 arrest, suggesting that the purpose of the delay is to give the cell time to repair damaged chromosomes before continuing with the cell cycle. [102] This ensures the proper functioning of mitosis.

                  Various species of animals exhibit similar mechanisms of cellular delay in response to DNA damage, which can be caused by exposure to x-irradiation. The budding yeast Saccharomyces cerevisiae has specifically been studied because progression through the cell cycle can be followed via nuclear morphology with ease. By studying Saccharomyces cerevisiae, researchers have been able to learn more about radiation-sensitive (RAD) genes, and the effect that RAD mutations may have on the typical cellular DNA damaged-induced delay response. Specifically, the RAD9 gene plays a crucial role in detecting DNA damage and arresting the cell in G2 until the damage is repaired.

                  Through extensive experiments, researchers have been able to illuminate the role that the RAD genes play in delaying cell division in response to DNA damage. When wild-type, growing cells are exposed to various levels of x-irradiation over a given time frame, and then analyzed with a microcolony assay, differences in the cell cycle response can be observed based on which genes are mutated in the cells. For instance, while unirradiated cells will progress normally through the cell cycle, cells that are exposed to x-irradiation either permanently arrest (become inviable) or delay in the G2 phase before continuing to divide in mitosis, further corroborating the idea that the G2 delay is crucial for DNA repair. However, rad strains, which are deficient in DNA repair, exhibit a markedly different response. For instance, rad52 cells, which cannot repair double-stranded DNA breaks, tend to permanently arrest in G2 when exposed to even very low levels of x-irradiation, and rarely end up progressing through the later stages of the cell cycle. This is because the cells cannot repair DNA damage and thus do not enter mitosis. Various other rad mutants exhibit similar responses when exposed to x-irradiation.

                  However, the rad9 strain exhibits an entirely different effect. These cells fail to delay in the G2 phase when exposed to x-irradiation, and end up progressing through the cell cycle unperturbed, before dying. This suggests that the RAD9 gene, unlike the other RAD genes, plays a crucial role in initiating G2 arrest. To further investigate these findings, the cell cycles of double mutant strains have been analyzed. A mutant rad52 rad9 strain—which is both defective in DNA repair and G2 arrest—fails to undergo cell cycle arrest when exposed to x-irradiation. This suggests that even if DNA damage cannot be repaired, if RAD9 is not present, the cell cycle will not delay. Thus, unrepaired DNA damage is the signal that tells RAD9 to halt division and arrest the cell cycle in G2. Furthermore, there is a dose-dependent response as the levels of x-irradiation—and subsequent DNA damage—increase, more cells, regardless of the mutations they have, become arrested in G2.

                  Another, and perhaps more helpful way to visualize this effect is to look at photomicroscopy slides. Initially, slides of RAD+ and rad9 haploid cells in the exponential phase of growth show simple, single cells, that are indistinguishable from each other. However, the slides look much different after being exposed to x-irradiation for 10 hours. The RAD+ slides now show RAD+ cells existing primarily as two-budded microcolonies, suggesting that cell division has been arrested. In contrast, the rad9 slides show the rad9 cells existing primarily as 3 to 8 budded colonies, and they appear smaller than the RAD+ cells. This is further evidence that the mutant RAD cells continued to divide and are deficient in G2 arrest.

                  However, there is evidence that although the RAD9 gene is necessary to induce G2 arrest in response to DNA damage, giving the cell time to repair the damage, it does not actually play a direct role in repairing DNA. When rad9 cells are artificially arrested in G2 with MBC, a microtubule poison that prevents cellular division, and then treated with x-irradiation, the cells are able to repair their DNA and eventually progress through the cell cycle, dividing into viable cells. Thus, the RAD9 gene plays no role in actually repairing damaged DNA—it simply senses damaged DNA and responds by delaying cell division. The delay, then, is mediated by a control mechanism, rather than the physical damaged DNA. [103]

                  On the other hand, it is possible that there are backup mechanisms that fill the role of RAD9 when it is not present. In fact, some studies have found that RAD9 does indeed play a critical role in DNA repair. In one study, rad9 mutant and normal cells in the exponential phase of growth were exposed to UV-irradiation and synchronized in specific phases of the cell cycle. After being incubated to permit DNA repair, the extent of pyrimidine dimerization (which is indicative of DNA damage) was assessed using sensitive primer extension techniques. It was found that the removal of DNA photolesions was much less efficient in rad9 mutant cells than normal cells, providing evidence that RAD9 is involved in DNA repair. Thus, the role of RAD9 in repairing DNA damage remains unclear. [104]

                  Regardless, it is clear that RAD9 is necessary to sense DNA damage and halt cell division. RAD9 has been suggested to possess 3’ to 5’ exonuclease activity, which is perhaps why it plays a role in detecting DNA damage. When DNA is damaged, it is hypothesized that RAD9 forms a complex with RAD1 and HUS1, and this complex is recruited to sites of DNA damage. It is in this way that RAD9 is able to exert its effects.

                  Although the function of RAD9 has primarily been studied in the budding yeast Saccharomyces cerevisiae, many of the cell cycle control mechanisms are similar between species. Thus, we can conclude that RAD9 likely plays a critical role in the DNA damage response in humans as well.


                  Lesson Objectives

                  • Name the types of asexual reproduction.
                  • Explain the advantage of sexual reproduction.
                  • List the stages of meiosis and explain what happens in each stage.

                  Check Your Understanding

                  • Can something that does not reproduce still be considered living?
                  • What stores the genetic information that is passed on to offspring?
                  • How many chromosomes are in the human nucleus?


                  • allele
                  • asexual reproduction
                  • binary fission
                  • crossing-over
                  • cross-pollination
                  • diploid
                  • external fertilization
                  • gamete
                  • gonad
                  • haploid
                  • internal fertilization
                  • meiosis
                  • ovaries
                  • parthenogenesis
                  • sexual reproduction
                  • testes
                  • zygote

                  What is reproduction?

                  What does reproduction mean? Can an organism be considered alive if it cannot make the next generation? Since individuals cannot live forever, they must reproduce for the species to survive. Reproduction is the ability to make the next generation.

                  Two methods of reproduction are:

                  1. Asexual reproduction, or the process of forming a new individual from a single parent.
                  2. Sexual reproduction, or the process of forming a new individual from two parents.

                  There are advantages and disadvantages to each method, but the result is always the same: a new life begins.

                  Asexual Reproduction

                  For humans to reproduce, DNA must be passed from the mother and father to the child. Humans cannot reproduce with just one parent, but it is possible in other organisms, like bacteria, some insects and some fish. These organisms can reproduce asexually, meaning that the offspring (children) have a single parent and share the exact same genetic material as the parent. This is very different from humans.

                  The advantage of asexual reproduction is that it can be very quick and does not require the meeting of a male and female organism. The disadvantage of asexual reproduction is that organisms cannot mix beneficial traits from both parents. An organism that is born through asexual reproduction only has the DNA from the one parent, and it is the exact copy of that parent. This can cause problems for the individual. For example, if the parent organism has a gene that causes cancer, the offspring will also have the gene that causes cancer. Organisms produced sexually may or may not inherit the cancerous gene because there are two parents mixing up their genes.

                  Types of organisms that reproduce asexually include:

                  1. Prokaryotic organisms, like bacteria. Bacteria reproduce through binary fission, where they grow and divide in half (Figurebelow). First, their chromosome replicates (bacteria only have one chromosome) and the cell enlarges. After cell division, the two new cells each have one identical chromosome (mitosis is not necessary because bacteria do not have nuclei). Then, new membranes form to separate the two cells. This simple process allows bacteria to reproduce very rapidly.
                  2. Flatworms, an animal species. Flatworms divide in two, then each half regenerates into a new flatworm identical to the original.
                  3. Different types of insects, fish, and lizards. These organisms can reproduce asexually through a process called parthenogenesis (Figurebelow). Parthenogenesis happens when an unfertilized egg cell grows into a new organism. The resulting organism has half the amount of genetic material of the parent. Parthenogenesis is common in honeybees. In a hive, the sexually produced eggs become workers, while the asexually produced eggs become drones.

                  Bacteria reproduce by binary fission. Shown is one bacterium reproducing and becoming two bacteria.

                  This Komodo dragon was born by parthenogenesis.

                  Sexual Reproduction

                  During sexual reproduction, two parents are involved. Most animals are dioecious, meaning there is a separate male and female sex, with the male producing sperm and the female producing eggs. When a sperm and egg meet, a zygote, the first cell of a new organism, is formed (Figure below). The zygote will divide and grow into the embryo.

                  During sexual reproduction, a sperm fertilizes an egg.

                  Let's explore how animals, plants, and fungi reproduce sexually:

                  • Animals often have gonads, organs that produce eggs or sperm. The male gonads are the testes, which produce the sperm, and the female gonads are the ovaries, which produce the eggs. Sperm and egg, the two sex cells, are known as gametes, and can combine two different ways:
                  1. Fish and other aquatic animals release their gametes in the water, which is called external fertilization. These gametes will combine by chance. (Figurebelow).
                  2. Animals that live on land reproduce by internal fertilization. Typically males have a penis that deposits sperm into the vagina of the female. Birds do not have penises, but they do have a chamber called the cloaca that they place close to another bird’s cloaca to deposit sperm.

                  This fish guards her eggs, which will be fertilized externally.

                  • Plants can also reproduce sexually, but their reproductive organs are different from animals’ gonads. Plants that have flowers have their reproductive parts in the flower. The sperm is contained in the pollen, while the egg is contained in the ovary, deep within the flower. The sperm can reach the egg two different ways:
                  1. In self-pollination, the egg is fertilized by the pollen of the same flower.
                  2. In cross-pollination, sperm from the pollen of one flower fertilizes the egg of another flower. Like other types of sexual reproduction, cross-pollination allows new combinations of traits. Cross-pollination occurs when pollen is carried by the wind to another flower. It can also occur when animal pollinators, like honeybees, or butterflies (Figurebelow) carry the pollen from flower to flower.
                  • Fungi can also reproduce sexually, but instead of female and male sexes, they have (+) and (-) strains. When the filaments of a (+) and (-) fungi meet, the zygote is formed. Just like in plants and animals, each zygote receives DNA from two parent strains.

                  Butterflies receive nectar when they deposit pollen into flowers, resulting in cross-pollination.

                  Meiosis and Gametes

                  Meiosis is a process of cell division that produces sex cells, or gametes. Gametes are reproductive cells, such as sperm and egg. As gametes are produced, the number of chromosomes must be reduced by half. Why? The zygote must contain information from the mother and from the father, so the gametes must contain half of the chromosomes found in normal body cells.

                  In humans, our cells have 23 pairs of chromosomes, and each chromosome within a pair is called a homologous chromosome. For each of the 23 chromosome pairs, you received one chromosome from your father and one chromosome from your mother. The homologous chromosomes are separated when gametes are formed. Therefore, gametes have only 23 chromosomes, not 23 pairs.

                  Alleles are alternate forms of genes found on chromosomes. Since the separation of chromosomes into gametes is random, it results in different combinations of chromosomes (and alleles) in each gamete. With 23 pairs of chromosomes, there is a possibility of over 8 million different combinations of chromosomes in a gamete.

                  Haploid vs. Diploid

                  A cell with two sets of chromosomes is diploid, referred to as 2n, where n is the number of sets of chromosomes. Most of the cells in a human body are diploid. A cell with one set of chromosomes, such as a gamete, is haploid, referred to as n. Sex cells are haploid. When a haploid sperm (n) and a haploid egg (n) combine, a diploid zygote will be formed (2n). In short, when a diploid zygote is formed, half of the DNA comes from each parent.


                  Before meiosis begins, DNA replication occurs, so each chromosome contains two sister chromatids that are identical to the original chromosome.

                  Meiosis is divided into two divisions: Meiosis I and Meiosis II. Each division is similar to mitosis and can be divided into the same phases: prophase, metaphase, anaphase, and telophase. Between the two divisions, DNA replication does not occur. Through this process, one diploid cell will divide into four haploid cells.

                  Meiosis I

                  During meiosis I, the pairs of homologous chromosomes are separated from each other.

                  1. Prophase I: The homologous chromosomes line up together. During this time, a process that only happens in meiosis can occur. This process is called crossing-over (Figurebelow), which is the exchange of DNA between homologous chromosomes. Crossing-over increases the new combinations of alleles in the gametes. Without crossing-over, the offspring would always inherit all of the many alleles on one of the homologous chromosomes. Also during prophase I, the spindle forms, the chromosomes condense as they coil up tightly, and the nuclear envelope disappears.
                  2. Metaphase I: The homologous chromosomes line up in pairs in the middle of the cell. Chromosomes from the mother or from the father can each attach to either side of the spindle. Their attachment is random, so all of the chromosomes from the mother or father do not end up in the same gamete. The gamete will contain some chromosomes from the mother and some chromosomes from the father.
                  3. Anaphase I: The homologous chromosomes separate.
                  4. Telophase I: The spindle fibers dissolves, but a new nuclear envelope does not need to form. This is because the nucleus will divide again. No DNA replication happens between meiosis I and meiosis II because the chromosomes are already duplicated.

                  During crossing-over, segments of DNA are exchanged between sister chromatids. Notice how this can result in an allele (A) on one sister chromatid being moved onto the other sister chromatid.

                  Meiosis II

                  During meiosis II, the sister chromatids are separated and the gametes are generated.

                  The steps are outlined below:

                  1. Prophase II: The chromosomes condense.
                  2. Metaphase II: The chromosomes line up one on top of the next along the middle of the cell.
                  3. Anaphase II: The sister chromatids separate.
                  4. Telophase II: Nuclear envelopes form around the chromosomes in all four cells.

                  After cytokinesis, each cell has divided again. Therefore, meiosis results in four daughter cells with half the DNA of the parent cell (Figure below). In human cells, the parent cell has 46 chromosomes, so the cells produced by meiosis have 23 chromosomes. These cells will become gametes.

                  Mitosis vs. Meiosis: A Comparison

                  Figure below is a comparison between binary fission, mitosis, and meiosis. Mitosis and meiosis are also compared in Table below.

                  A comparison between binary fission, mitosis, and meiosis.

                  Animations of meiosis can be found at the following sites:

                  Lesson Summary

                  • Organisms can reproduce sexually or asexually.
                  • The gametes in sexual reproduction must have half the DNA of the parent.
                  • Meiosis is the process of nuclear division that forms gametes.

                  Review Questions


                  2. During what phase of meiosis do homologous chromosomes separate?

                  3. What is the purpose of meiosis?

                  4. In what phase of meiosis do homologous chromosomes pair up?

                  Apply Concepts

                  5. Explain how organisms reproduce asexually.

                  6. Explain how birds fertilize their eggs.

                  7. How do most plants reproduce sexually?

                  8. Compare and contrast the process of mitosis and the process of meiosis.

                  Critical Thinking

                  9. How would sexual reproduction in a lizard be different than in a fish?

                  10. What is the advantage of sexual reproduction over asexual reproduction?

                  11. If an organism has 12 chromosomes in its cells, how many chromosomes will be in its gametes?

                  Further Reading / Supplemental Links

                  Points to Consider

                  • What must be replicated prior to mitosis?
                  • How do you think DNA might be replicated?
                  • What might happen if there is a mistake during DNA replication?

                  Why is DNA replication not 100% accurate - Biology

                  DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations in other cases, repair enzymes are themselves mutated or defective.

                  Most of the mistakes during DNA replication are promptly corrected by DNA polymerase by proofreading the base that has just been added (Figure 1). In proofreading, the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the exonuclease action of DNA pol III. Once the incorrect nucleotide has been removed, a new one will be added again.

                  Figure 1. Proofreading by DNA polymerase corrects errors during replication.

                  Some errors are not corrected during replication, but are instead corrected after replication is completed this type of repair is known as mismatch repair (Figure 2). The enzymes recognize the incorrectly added nucleotide and excise it this is then replaced by the correct base. If this remains uncorrected, it may lead to more permanent damage. How do mismatch repair enzymes recognize which of the two bases is the incorrect one? In E. coli, after replication, the nitrogenous base adenine acquires a methyl group the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, DNA polymerase is able to remove the wrongly incorporated bases from the newly synthesized, non-methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has completed.

                  Figure 2. In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base.

                  In another type of repair mechanism, nucleotide excision repair, enzymes replace incorrect bases by making a cut on both the 3′ and 5′ ends of the incorrect base (Figure 3).

                  Figure 3. Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

                  The segment of DNA is removed and replaced with the correctly paired nucleotides by the action of DNA pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.

                  Why is DNA replication not 100% accurate - Biology

                  Your DNA contains a record of your ancestors, but you aren't a carbon copy of any one of them. The particular mix of DNA you inherit is unique to you. You receive 50% of your DNA from each of your parents, who received 50% of theirs from each of their parents, and so on. In the chart below you can see how the amount of DNA you receive from a particular ancestor decreases over generations. If you go back far enough, there is a chance that you inherited no DNA from a particular ancestor. The chart below helps illustrate how different segments of DNA might have been passed down from your grandparents to make your unique DNA. Assume each letter represents a segment of DNA. Things to notice are:

                  • Which letters get passed down to each generation is random (the fact that the letters spell names in this example is simply to help with the illustration).
                  • Not all of the letters get passed down.
                  • Just because a child doesn't have a letter doesn't mean that an earlier ancestor didn't have that letter.
                  • Siblings can have different combinations of letters

                  In the example on the chart, your paternal grandfather has the unique DNA of ANDREW. He can pass down only 50% of his DNA to each child. In your father's case, the pieces of DNA' randomly selected to be passed on to him are represented by the letters DEW. At the same time, grandmother SANDRA provides the randomly selected segments ADR, which combine with her husband's DEW to create your father's unique genetic signature: EDWARD. Notice that not all of the letters from ANDREW and SANDRA get passed down to EDWARD. Your father, EDWARD, has three children with your mother, whose genetic signature is ANGELA. EDWARD and ANGELA each pass 50% of their DNA, randomly selected, to each of their children, who end up with the genetic signatures GLENDA, GERALD, and REAGAN. Again, the parents don't get to choose which segments (letters) go to each child. And while having more children increases the chances of passing on more of your DNA, if you look closely, you'll see that even with three children, not all of EDWARD and ANGELA's DNA segments made it to the next generation. This is a simplified example of how genetic inheritance works in all of us. By understanding how DNA is inherited, you can see how and why you have some DNA segments that match your relatives, and others that do not, why you may or may not have inherited DNA segments associated with a certain ethnicity, and why getting multiple people in your family tested can help discover more of your family's genetic tree. I have had fun learning about my own DNA inheritance, especially after I had a few of my family members tested. Below are 4 sets of DNA ethnicity results from me and my three siblings. Our results are a great example of how genetic inherence is random, just like the letter block example above explains. Do you see how different we are' Focus in on the Europe West ethnicity region between us. If you look at the results on the far right, (which happen to be mine) European West almost doesn't exist. If fact, I would say, in comparison to my sibling's results Europe West isn't being represented at all in my results. But my oldest sibling, (far left) has 32%, next sibling has 5%, and the third one has 16%. My sibling's DNA results are all different-because we are all different. None of us are twins so we expect our results to be different in some ways. Genetic inheritance is random and my sibling's ethnicity results are a great example of that. But because our ethnicity results are different doesn't mean we aren't siblings. We all show up as immediate' family in the matching section which is expected. This is why it's important and fun to get others in your family tested. Each of us carry unique pieces of DNA that can unlock our family's story. If I had just used my DNA results to infer my genetic story, I would have missed out on a few pieces. So it's important to get parents, siblings, aunts, uncles and even 1 st cousins tested to help you do more with your DNA results. Click here to get others tested. To learn even more about DNA inheritance and how AncestryDNA determines genetic ethnicity click here.

                  Watch the video: DNA Replication Updated (August 2022).