9: Details of DNA Replication and Repair - Biology

9: Details of DNA Replication and Repair - Biology

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  • 9.1: Introduction
    Replication begins at one or more origins of replication along DNA, where helicase enzymes catalyze unwinding of the double helix. DNA unwinding creates replicating bubbles, or replicons, with replication forks at either end. Making a new DNA strand starts with making an RNA primer with RNA nucleotides and primase enzymes.
  • 9.2: DNA Replication
    As we’ve seen, DNA strands have directionality, with a 5’ nucleotide-phosphate and a 3’ deoxyribose hydroxyl end. This is even true for circular bacterial chromosomes…, if the circle is broken! Because the strands of the double helix are antiparallel, the 5’ end of one strand aligns with the 3’end of the other at both ends of the double helix. The complementary pairing of bases in DNA means that the base sequence of one strand can be used as a template to make a new complementary strand. As we’l
  • 9.3: DNA Repair
    We generally accept the notion that replication faithfully duplicates the genetic material. At the same time, evolution would not be possible without mutation, and mutation is not possible without at least some adverse consequences. Germline mutations are heritable. When present in one, but especially in both alleles of a gene, such mutations can result in genetic disease (e.g., Tay-Sach’s disease, cystic fibrosis, hemophilia, sickle-cell anemia, etc.).
  • 9.4: Key Words and Terms

Causes and consequences of replication stress

Replication stress is a complex phenomenon that has serious implications for genome stability, cell survival and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATR (ATM- and Rad3-related). Along with its downstream effectors, ATR stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding this response may be key to diagnosing and treating human diseases caused by defective responses to replication stress.

Sources of DNA Damage

For many years, exogenous sources of damage have been thought to be the primary cause of DNA mutations leading to cancer. However, Jackson and Loeb proposed that endogenous sources of DNA damage also contribute significantly to mutations that lead to malignancy. 5 Both environmental and cellular sources can result in similar types of DNA damage.

DNA can be attacked by physical and chemical mutagens. Physical mutagens are primarily radiation sources, including UV (200-300 nm wavelength) radiation from the sun. UV radiation produces covalent bonds that crosslink adjacent pyrimidine (cytosine and thymine) bases in the DNA strand. Ionizing radiation (X-rays) initiates DNA mutations by generating free radicals within the cell that create reactive oxygen species (ROS) and result in single-strand and double-strand breaks in the double helix. Chemical mutagens can attach alkyl groups covalently to DNA bases nitrogen mustard compounds that can methylate or ethylate the DNA base are examples of DNA alkylating agents. Procarcinogens are chemically inert precursors that are metabolically converted into highly reactive carcinogens. These carcinogens can react with DNA by forming DNA adducts, i.e., chemical entities attached to DNA. Benzo[a]pyrene, a polyaromatic heterocycle, is not itself carcinogenic. It undergoes two sequential oxidation reactions mediated by cytochrome P450 enzymes, which results in benzo[a]pyrenediol epoxide (BPDE), the carcinogenic metabolite that is able to form a covalent DNA adduct (Figure 2).

Figure 2. Benzo[a]pyrene is oxidized by P450 enzymes to create the highly carcinogenic benzo[a]pyrenediolepoxide.

DNA damage can also result from endogenous metabolic and biochemical reactions, some of which are not well understood. 6 Hydrolysis reactions can partially or completely cleave the nucleotide base from the DNA strand. The chemical bond connecting a purine base (adenine or guanine) to the deoxyribosyl phosphate chain can spontaneously break in the process known as depurination. An estimated 10,000 depurination events occur per day in a mammalian cell. 7 Depyrimidination (loss of pyrimidine base from thymine or cytosine) also occurs, but at a rate 20 to 100-fold lower than depurination.

Deamination occurs within the cell with the loss of amine groups from adenine, guanine, and cytosine rings, resulting in hypoxanthine, xanthine, and uracil, respectively. DNA repair enzymes are able to recognize and correct these unnatural bases. However, an uncorrected uracil base may be misread as a thymine during subsequent DNA replication and generate a C→T point mutation.

DNA methylation, a specific form of alkylation, occurs within the cell due to a reaction with S-adenosyl methionine (SAM). SAM is an intracellular metabolic intermediate that contains a highly reactive methyl group. In mammalian cells, methylation occurs at the 5-position of the cytosine ring of a cytidine base (C) that is 5’ to a guanosine base (G), i.e., sequence CpG. A significant source of mutation error is the spontaneous deamination of the 5-methylcytosine product of methyl-ation. Loss of the amine group results in a thymine base, which is not detected by DNA repair enzymes as an unnatural base. The resulting substitution is retained in DNA replication, creating a C→T point mutation (Figure 3).

Figure 3. The 2-phase mutation of cytosine results in thymine, creating a C→T point mutation.

Normal metabolic processes generate reactive oxygen species (ROS), which modify bases by oxidation. Both purine and pyrimidine bases are subject to oxidation. The most common mutation is guanine oxidized to 8-oxo-7,8-dihydroguanine, resulting in the nucleotide 8-oxo-deoxy guano sine (8-oxo-dG). The 8-oxo-dG is capable of base pairing with deoxyadenosine, instead of pairing with deoxycytotidine as expected. If this error is not detected and corrected by mismatch repair enzymes, the DNA subsequently replicated will contain a C→A point mutation. ROS may also cause depurination, depyrimidination, and single-strand or double strand breaks in the DNA.

Other genomic mutations may be introduced during DNA replication in the S phase of the cell cycle. Polymerases that duplicate template DNA have a small but significant error rate, and may incorporate an incorrect nucleotide based on Watson-Crick pairing versus the template DNA. Chemically altered nucleotide precursors may be incorporated into the generated DNA by the polymerase, instead of normal bases. In addition, polymerases are prone to “stuttering” when copying sections of DNA that contain a large number of repeating nucleotides or repeating sequences (microsatellite regions). This enzymatic “stuttering” is due to a strand slippage, when the template and replicated strands of DNA slip out of proper alignment. As a result, the polymerase fails to insert the correct number of nucleotides indicated by the template DNA, resulting in too few or too many nucleotides in the daughter strand.

Single strand and double strand cleavage of the DNA may occur. Single strand breaks may result from damage to the deoxyribose moiety of the DNA deoxyribosylphosphate chain. Breaks also result as an intermediate step of the base excision repair pathway after the removal of deoxyribose phosphate by AP-endonuclease 1. 8 When a single strand break occurs, both the nucleotide base and the deoxyribose backbone are lost from the DNA structure. Double strand cleavage most often occurs when the cell is passing through S-phase, as the DNA may be more susceptible to breakage while it is unraveling for use as a template for replication.

14.6 DNA Repair

By the end of this section, you will be able to do the following:

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 the proofreading ability of DNA polymerase itself. (Figure 14.17). 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 3' exonuclease action of DNA pol. Once the incorrect nucleotide has been removed, it can be replaced by the correct one.

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 14.18). Specific repair enzymes recognize the mispaired nucleotide and excise part of the strand that contains it the excised region is then resynthesized. If the mismatch remains uncorrected, it may lead to more permanent damage when the mismatched DNA is replicated. 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.

Another type of repair mechanism, nucleotide excision repair , is similar to mismatch repair, except that it is used to remove damaged bases rather than mismatched ones. The repair enzymes replace abnormal bases by making a cut on both the 3' and 5' ends of the damaged base (Figure 14.19). 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.

A well-studied example of mistakes not being corrected is seen in people suffering from xeroderma pigmentosa (Figure 14.20). Affected individuals have skin that is highly sensitive to UV rays from the sun. When individuals are exposed to UV light, pyrimidine dimers, especially those of thymine, are formed people with xeroderma pigmentosa are not able to repair the damage. These are not repaired because of a defect in the nucleotide excision repair enzymes, whereas in normal individuals, the thymine dimers are excised and the defect is corrected. The thymine dimers distort the structure of the DNA double helix, and this may cause problems during DNA replication. People with xeroderma pigmentosa may have a higher risk of contracting skin cancer than those who don't have the condition.

Errors during DNA replication are not the only reason why mutations arise in DNA. Mutations , variations in the nucleotide sequence of a genome, can also occur because of damage to DNA. Such mutations may be of two types: induced or spontaneous. Induced mutations are those that result from an exposure to chemicals, UV rays, x-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent they are a result of natural reactions taking place within the body.

Mutations may have a wide range of effects. Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These substitutions can be of two types, either transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind for example, a purine such as adenine may be replaced by the purine guanine. Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa for example, cytosine, a pyrimidine, is replaced by adenine, a purine. Some point mutations are not expressed these are known as silent mutations. Silent mutations are usually due to a substitution in the third base of a codon, which often represents the same amino acid as the original codon. Other point mutations can result in the replacement of one amino acid by another, which may alter the function of the protein. Point mutations that generate a stop codon can terminate a protein early.

Some mutations can result in an increased number of copies of the same codon. These are called trinucleotide repeat expansions and result in repeated regions of the same amino acid. Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a base, also known as deletion. If an insertion or deletion results in the alteration of the translational reading frame (a frameshift mutation), the resultant protein is usually nonfunctional. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome this is also known as translocation. These mutation types are shown in Figure 14.21.

Visual Connection

A frameshift mutation that results in the insertion of three nucleotides is often less deleterious than a mutation that results in the insertion of one nucleotide. Why?

Mutations in repair genes have been known to cause cancer. Many mutated repair genes have been implicated in certain forms of pancreatic cancer, colon cancer, and colorectal cancer. Mutations can affect either somatic cells or germ cells. If many mutations accumulate in a somatic cell, they may lead to problems such as the uncontrolled cell division observed in cancer. If a mutation takes place in germ cells, the mutation will be passed on to the next generation, as in the case of hemophilia and xeroderma pigmentosa.

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