DNA Repair Pathways PDF

Molecular Biology and Cytogenetics 6. DNA Repair Pathways Instructor: Dr. Mohammad Abukhalil Error occurrence in DNA replication ⚫ Initial pairing errors between incoming nucleotides and those in the template strand occur at a rate of one in 105 nucleotides. ⚫ However, errors in...

Molecular Biology and Cytogenetics 6. DNA Repair Pathways Instructor: Dr. Mohammad Abukhalil Error occurrence in DNA replication ⚫ Initial pairing errors between incoming nucleotides and those in the template strand occur at a rate of one in 105 nucleotides. ⚫ However, errors in the completed DNA molecule amount to only one in 1010 (10 billion) nucleotides, an error rate that is 100,000 times lower. ⚫ That’s because they are usually detected and fixed by DNA proofreading and repair mechanisms. ⚫ DNA replication errors lead to a permanent mutation in the next round of DNA replication if not repaired. Proofreading ⚫ Proofreading is an error-correcting reaction involved in DNA replication and other processes. ⚫ During DNA replication, DNA polymerases proofread each nucleotide against its template as soon as it is covalently bonded to the growing strand. ⚫ Upon finding an incorrectly paired nucleotide, the polymerase removes the nucleotide and then resumes synthesis.  Mismatch repair system ⚫ A DNA mismatch repair system removes replication errors that escape proofreading. ⚫ Mismatch repair mainly fixes mispaired bases right after DNA replication. ⚫ A complex of mismatch repair proteins recognizes such a DNA mismatch, removes a portion of the DNA strand containing the error, and then resynthesizes the missing DNA. When two bases are mispaired, which of the DNA strands contains the error? ⚫ In bacteria, newly synthesized DNA lacks a type of chemical modification called methylation that is present on the preexisting parent DNA. ⚫ An old DNA strand will have methyl (−CH₃) groups attached to some of its bases, while a newly made DNA strand will not yet have gotten its methyl group. ⚫ In eukaryotes, the processes that allow the original strand to be identified in mismatch repair involve recognition of nicks (single- stranded breaks) that are found only in the newly synthesized DNA. Mismatch repair in human disease ⚫ Hereditary nonpolyposis colorectal cancer (also called Lynch syndrome) is caused by mutations in genes encoding certain mismatch repair proteins. ⚫ Since mismatched bases are not repaired in the cells of people with this syndrome, mutations accumulate much more rapidly than in the cells of an unaffected person. This can lead to the development of tumors in the colon. General classes of DNA damage General classes of DNA damage ⚫ Spontaneous damage to DNA can occur through the action of water in the aqueous environment of the cell. ⚫ A mutagen is any chemical agent that causes an increase in the rate of mutation above the spontaneous background. Three general classes of DNA damage ⚫ Single base changes ⚫ Structural distortion ⚫ DNA backbone damage  Single base changes ⚫ A single base change or “conversion” affects the DNA sequence but has only a minor effect on overall structure. ⚫ Deamination is the most frequent and important kind of hydrolytic damage. ⚫ Methylated cytosines are “hotspots” for spontaneous mutation in vertebrate DNA because deamination of 5-methylcytosine generates thymine. ⚫ Alkylating agents such as nitrosamines lead to the formation of O6-methylguanosine. ⚫ This modified base often mispairs with thymine. ⚫ Can result in a GC→GT→AT point mutation after DNA replication.  Structural distortion ⚫ UV radiation induces that formation of a cyclobutane ring between adjacent thymines, forming a T-T dimer. ⚫ The T-T dimer distorts the double helix and can block transcription and replication. ⚫ UV radiation can also induce dimers between cytosine and thymine. ⚫ Other bulky adducts can be induced by chemical mutagenesis. ⚫ Structural distortion can be caused by intercalating agents and base analogs: ⚫ Ethidium bromide has several flat polycyclic rings that insert between the DNA bases. ⚫ 5-bromouracil, an analog of thymine, can mispair with guanine. DNA backbone damage Formation of abasic sites ⚫ Loss of the nitrogenous base from a nucleotide. ⚫ Generated spontaneously by the formation of unstable base adducts. Double-stranded DNA breaks ⚫ Induced by ionizing radiation and a wide range of chemical compounds. ⚫ The most severe type of DNA damage. Cellular responses to DNA damage ⚫ Damage bypass ⚫ Damage reversal ⚫ Damage removal Lesion bypass Translesion synthesis (TLS) ⚫ Specialized low-fidelity, “error-prone” DNA polymerases transiently replace the replicative polymerases and copy past damaged DNA. ⚫ Typical error rates range from 10-1 to 10-3 per base pair. DNA polymerase eta () ⚫ Performs translesion synthesis past TT dimers by inserting AA. DNA damage repair mechanisms ⚫ Although DNA is a highly stable material—as required for the storage of genetic information—it is a complex organic molecule that is susceptible, even under normal cell conditions, to spontaneous changes that would lead to mutations if left unrepaired. ⚫ Spontaneous alterations that require DNA repair include oxidative damage, hydrolytic attack and methylation. ⚫ There are many repair processes that help fix damaged DNA, including direct reversal of damage, excision repair and double- stranded break repair. Direct reversal of DNA damage Reversal of thymine-thymine dimers by DNA photolyase ⚫ In most organisms, UV radiation damage to DNA can be directly repaired. ⚫ DNA photolyase uses energy from near UV to blue light to break the covalent bonds holding two adjacent pyrimidines together. Damage reversal by DNA methyltransferase ⚫ Guanine (G) can undergo a reaction that attaches a methyl (−CH₃) group to an oxygen atom in the base. The methyl- bearing guanine, if not fixed, will pair with thymine (T) rather than cytosine (C) during DNA replication. ⚫ Methyltransferase catalyzes the transfer of the methyl group on O6-methylguanine to the sulfhydryl group of a cysteine residue on the enzyme.  Repair of single base changes and structural distortions by removal of DNA damage Excision repair ⚫ Damage to one or a few bases of DNA is often fixed by removal (excision) and replacement of the damaged region. ⚫ In base excision repair, just the damaged base is removed. ⚫ In nucleotide excision repair, a patch of nucleotides is removed. Base excision repair ⚫ It is a mechanism used to detect and remove certain types of damaged bases. ⚫ A group of enzymes called DNA glycosylases play a key role in base excision repair. Each glycosylase detects and removes a specific kind of damaged base by its hydrolytic removal. ⚫ Depurination and deamination are the most frequent chemical reactions that are repaired by base excision mechanism. Depurination and deamination ⚫ Deamination can convert a cytosine base into uracil, a base typically found only in RNA. During DNA replication, uracil will pair with adenine rather than guanine, so an uncorrected cytosine-to-uracil change can lead to a mutation. ⚫ Depurination involves the loss of purine bases (adenine and guanine) from DNA, producing an abasic site. Base excision mechanism ⚫ DNA glycosylase finds the damaged base that it recognizes and removes that base from its sugar. ⚫ The “missing tooth” created by DNA glycosylase action is recognized by an enzyme called AP endonuclease. ⚫ AP endonuclease and phosphodiesterase remove sugar phosphate. ⚫ The gap is filled and sealed by other enzymes (DNA polymerase and DNA ligase). Nucleotide excision repair ⚫ It is another pathway used to remove and replace damaged bases. ⚫ This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. ⚫ Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene, found in tobacco smoke) as well as the various pyrimidine dimers, such as thymine dimer caused by sunlight. Nucleotide excision mechanism ⚫ In this pathway, a large multienzyme complex scans the DNA for a distortion in the double helix. ⚫ Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. ⚫ The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase Xeroderma pigmentosum ⚫ People with xeroderma pigmentosum are extremely sensitive to UV light. This condition is caused by mutations affecting the nucleotide excision repair pathway. When this pathway doesn't work, thymine dimers and other forms of UV damage can't be repaired. ⚫ People with xeroderma pigmentosum develop severe sunburns from just a few minutes in the sun, and about half will get skin cancer by the age of 10 unless they avoid the sun. Double-strand break repair by removal of DNA damage Double-strand breaks are efficiently repaired ⚫ When damage from ionizing radiation, oxidative free radicals, or chemotherapeutic agents causes both the strands of DNA to be broken. ⚫ If these were left unrepaired, they would quickly lead to the breakdown of chromosomes into smaller fragments and to loss of genes when the cell divides. ⚫ However, two distinct mechanisms have evolved to correct the damage, homologous recombination and nonhomologous end joining. Homologous recombination ⚫ Repairs double-strand breaks by retrieving genetic information from an undamaged homologous chromosome. ⚫ It occurs in newly replicated DNA (in S and G2 phases). ⚫ Here, the DNA is repaired using the unaffected sister chromatid as a template for proper repair of breaks. ⚫ Fanconi’s anemia is a condition caused by failures in DNA recombination repair enzymes to correct the defects by homologous recombination. Nonhomologous end joining ⚫ The two broken ends of the chromosome are simply brought together and rejoined by DNA ligation, generally with the loss of nucleotides at the site of joining ⚫ It is error prone since it can also introduce mutations during repair. ⚫ Nonhomologous end joining is especially important before the cell has replicated its DNA because there is no template available for repair by homologous recombination. DNA damage delays progression of the cell cycle ⚫ Because of the importance of maintaining intact, undamaged DNA from generation to generation, eukaryotic cells have an additional mechanism that maximizes the effectiveness of their DNA repair enzymes. ⚫ They delay progression of the cell cycle until DNA repair is complete. ⚫ In mammalian cells, the presence of DNA damage can block entry from G1 into S phase, it can slow S phase once it has begun, and it can block the transition from G2 phase to M phase.