| Cells can repair damaged DNA
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| Because DNA is the reservoir of genetic information within the cell, it is extremely important to maintain the integrity of DNA. Therefore, the cell has developed highly efficient mechanisms for the repair of modified or damaged DNA.
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| DNA is subject to numerous types of endogenous and environmental insults that cause nucleotide deletions, insertions, sequence inversions and transpositions. Some of this damage is secondary to chemical modification of DNA by alkylating agents (including many carcinogens), reactive oxygen
species (Chapter 35) and ionizing radiation (ultraviolet or radioactive). Both the sugar and bases of DNA are subject to modification, yielding an estimated 10 000 to 100 000 modifications of DNA per cell per day. The nature of this damage is quite variable, including modification of single bases, single or double-strand breaks, and crosslinking between bases or bases and proteins. Oxidative damage is probably the most common form of DNA damage; it is increased in inflammation, by smoking, in aging and in age-related diseases, including atherosclerosis, diabetes and neurodegenerative diseases (Chapter 42). If not repaired, the accumulated damage will lead to permanent changes in the structure of DNA, setting the stage for loss of cellular functions, cell death, or cancer.
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| Numerous chemical and environmental agents are known that produce specific chemical modification of the nucleotides in the DNA strand, leading to mismatches during DNA synthesis. After chromosomal replication, the resulting daughter strand contains a different DNA sequence (mutation) from the parent strand. Cells use excision repair to remove alkylated nucleotides and other unusual base analogs, thereby protecting the DNA sequence from mutations. The unmodified strand serves as the template for the repair process.
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| When short-wavelength ultraviolet (UV) light interacts with DNA, adjacent thymine bases undergo an unusual dimerization, producing a cyclobutylthymine dimer in the DNA strand (Fig. 30.8). The primary mechanism for repair of these intra-strand thymine dimers is an excision repair mechanism. An endonuclease, which appears to be specific for this type of modification, cleaves the dimer-containing strand near the thymine dimer, and a small portion of that strand is removed. DNA polymerase I, the same enzyme that is involved in DNA biosynthesis, then recognizes and fills in the resulting gap. DNA ligase completes the repair by rejoining the DNA strands.
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Those nucleotides that contain amines, cytosine and adenosine , may spontaneously deaminate to form uracil or hypoxanthine, respectively. When these bases are found in DNA, specific N-glycosylases remove them. This produces base-pair gaps that are recognized by specific apurinic or apyrimidinic endonucleases that cleave the DNA near the site of the defect. An exonuclease then removes the stretch of the DNA strand containing the defect. A repair DNA polymerase replaces the DNA, and, finally, DNA ligase rejoins the DNA strand. This repair mechanism is referred to as excision repair.
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| Figure 30.8 Thymine dimer. A thymine dimer consists of a cyclobutane ring joining a pair of adjacent thymine nucleotides. |
| Single base-pair alterations also include depurination. The purine-N-glycosidic bonds are especially labile, so that an estimated
3-7 purines are removed from DNA per min per cell. Specific enzymes recognize these depurinated sites, and the base is replaced without interruption of the phosphodiester backbone.
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| Single-stranded breaks are frequently induced by ionizing radiation. These are repaired by direct ligation or by excision repair mechanisms. Double-stranded breaks are produced by ionizing radiation and some chemotherapeutic agents. Otherwise, double-stranded ends of DNA are rare in vivo; they are found at the end of chromosomes and in some specialized complexes involved in gene rearrangement. A specialized enzyme system is designed to recognize and rejoin these ends, but if the ends drift away from one another, the damage is not readily repaired.
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| Figure 30.9 Oxidative damage to DNA. 8-Oxo-2'-deoxyguanosine (8-oxoG) is an oxidative modification of DNA that causes mutations in the DNA strand. |
| Errors that escape the proof-reading activity of DNA polymerase III appear in newly synthesized DNA in the form of nucleotide mismatches. While readily repairable, the critical issue is identification of the strand to be repaired: which nucleotide strand is the daughter strand containing the error? In bacterial systems, mismatch repair is accomplished by methylation of DNA at adenine residues in specific sequences spaced along the genome; methylation does not affect base pairing. Newly synthesized strands lack methylated adenine residues, so that the mismatch repair system
enzymes scan the DNA, identify the mismatch, and then repair the unmethylated strand by excision repair. A similar approach is used to correct mismatches occurring during synthesis of mammalian DNA. Defects in mismatch repair are associated with hereditary nonpolyposis colon cancer, an autosomal dominant condition in humans.
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| Mutagens are chemical compounds that induce changes in the DNA sequence. A large number of natural and man-made chemicals are mutagenic. To evaluate the potential to mutate DNA, the American biochemist Bruce Ames developed a simple test, using special Salmonella typhimurium strains that cannot grow in the absence of histidine (His- phenotype). These histidine auxotrophic strains contain nucleotide substitutions or deletions that prevent the production of histidine biosynthetic enzymes. |
| To test for mutagenesis, about 10-9 mutant bacteria are spread on a culture plate lacking histidine. The suspected mutagen is added to the bacteria. The action of the mutagen occasionally results in the reversal of the histidine mutation, yielding a revertant strain that can now synthesize histidine and will grow in its absence. The mutagenicity of a compound is scored by counting the number of colonies that have reverted to the His+ phenotype. There is a good correlation between results of the Ames mutagenicity test and direct tests of carcinogenic activity in animals. |
| Some chemicals (procarcinogens) are not mutagenic per se, but are activated to mutagenic compounds during metabolic processes, e.g. during drug detoxification in liver or kidney. Benzopyrene, for example, is not mutagenic, but during its detoxification in liver, it is converted to diolepoxides which are potent mutagens and carcinogens. To provide sensitivity for detecting these compounds, the culture medium is supplemented with an extract of liver microsomes. |
| About 20 different oxidative modifications of DNA have been characterized; the most studied is 8-oxo-2'-deoxyguanosine (8-oxoG) (Fig. 30.9). During the process of DNA replication, mismatches between the modified 8-oxoG nucleoside in the template strand and incoming nucleotide triphosphates result in G-to-T transversions, thereby introducing mutations into the DNA strand. Although excision repair mechanisms
are effective, 8-oxoG, like other modified bases, may be reincorporated into DNA following excision.
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| Recently a mammalian protein, MTH1, was characterized that specifically degrades 8-oxo-dGTP, thereby preventing misincorporation of this altered nucleotide into DNA. Gene targeting was used to develop an MTH1-knockout mouse. Compared to the wild-type animal, the knockout showed a greater number of tumors in lung, liver, and stomach, illustrating the importance of post-repair protection mechanisms.
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| In lung cells, inhalation of some particulate materials results in an increase in 8-oxoG levels. The inflammatory process may play a role in asbestos-induced formation of lung tumors. Smoking also induces oxidative damage and increases levels of DNA oxidation products in lungs, blood and urine.
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