Biochemistry 201: DNA repair January 24, 26, 2000 Gilbert Chu

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1 1) Why study DNA repair? Biochemistry 201: DNA repair January 24, 26, 2000 Gilbert Chu The genome is assaulted by a myriad of different agents that cause DNA damage. Sources of damage can be both exogenous and endogenous. Exogenous sources include ultraviolet radiation from the sun, ionizing radiation from the radon decay, aflatoxin from fungus, burning tobacco, and many chemotherapy drugs. Endogenous sources include oxidative metabolism, spontaneous alterations of DNA, and V(D)J recombination, the mechanism that generates immunological diversity. Many mechanisms employing different biochemical strategies repair the many forms of DNA damage. In fact, DNA repair genes account for a significant fraction of the genome. DNA repair defects lead to genetic instability, which is a hallmark of cancer. Cancer occurs only when a cell accumulates mutations in several different genes, e.g. 6 or 7 in the case of colon cancer. Cancers accumulate this many mutations only after acquiring a mutation that promotes to genetic instability. In fact, it is this genetic instability that allows cancer cells to become resistant to treatment and underlies our failure to cure most cancers. 2) DNA double-strand break repair Sources of DNA double-strand breaks include: ionizing radiation, topoisomerase inhibitors such as etoposide and adriamycin, and V(D)J recombination.

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4 Breast cancer and double-strand break repair Breast cancer is linked to ionizing radiation exposure women exposed to atomic bombs over Hiroshima & Nagasaki women treated with ionizing radiation for post-partum mastitis mice exposed to ionizing radiation Familial breast/ovarian cancer is caused by mutation of the Brca1 or Brca2 genes. Brca1 and Brca2 proteins interact with Rad51 (the eukaryotic RecA homolog). Homozygous mutation of Brca2 causes sensitivity to ionizing radiation. Heterozygous mutation of Brca2 causes intermediate sensitivity to ionizing radiation. a) Nonhomologous end-joining Nonhomologous end-joining is a biochemical pathway for joining DNA ends without the benefit of an homologous chromosome. Because broken DNA ends may not be ligatable, this pathway often involves the loss of genetic information. Defects in nonhomologous end-joining lead to both ionizing radiation sensitivity and severe combined immunodeficiency. Double-strand break repair genes for nonhomologous end-joining Genes Proteins Protein function XRCC5, XRCC6 Ku86 / Ku70 binds to DNA ends and recruits DNA-PK CS XRCC7 DNA-PK CS DNA-dependent protein kinase: ser/thr kinase activated by DNA ends XRCC4, ligase IV XRCC4 / ligase IV dimer with DNA ligation activity 4

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7 Eukaryotes contain homologs of the E. coli mut proteins. Although humans also methylate their DNA, yeast and Drosophila do not. In humans and other eukaryotes, the mechanism for strand specificity remains unknown. Human mismatch repair proteins Protein Heterodimer Repair function MutSα MSH2 MSH6 binds base-base mismatches and insertion-deletion mismatches MutSβ MSH2 MSH3 binds insertion-deletion mismatches MutLα MLH1 PMS2 early step before excision Clinical: Genetics: Hereditary nonpolyposis colon cancer (HNPCC) Susceptibility to colon cancer and to other gastrointestinal cancers and endometrial cancer Autosomal dominant (affects 1/200 individuals) Patients are heterozygous for mutations in mismatch repair genes Tumors exhibit microsatellite instability (MIN+) 3) Mechanisms for repairing base damage Damage to DNA bases can be repaired by three major mechanisms: direct reversal of the damage, base excision repair (removal of the damaged base), and nucleotide excision repair (removal of an oligonucleotide containing the damaged base). 7

8 a) Direct reversal O6 methylguanine methyltransferase (O6MGT) provides one example of how DNA damage can be reversed directly. Cells contain methylation pathways that can accidentally methylate guanine to form O6-methylguanine, which can pair with thymine during replication and is therefore mutagenic. O6-methylguanine is recognized by O6MGT, which as its name implies, transfers the methyl group from guanine to its own cysteine 321. O6MGT is also capable of transferring the alkyl group from a phosphotriester on DNA to its own cysteine 69. Thus, O6MGT commits molecular suicide, becoming irreversibly inactivated, rather than acting as a true enzyme. a) Base excision repair Spontaneous deamination can lead to base mispairing and thus mutations that are fixed during DNA replication. For example, deamination converts cytosine to uracil, which would then be paired with adenine during replication. Uracil N-glycosylase recognizes and removes the uracil base 8

9 b) Removal of AP sites AP (apurinic/apyrimidinic) sites are DNA sites in which the base has been lost spontaneously or removed by the action of a glycosylase. A mammalian cell loses about 10,000 purines and 500 pyrimidines per day. AP endonucleases hydrolyze the phosphodiester bond 5' to the AP site to initiate the removal of the abasic deoxyribose for replacement by the proper nucleotide. c) Nucleotide excision repair (NER) This is a strategy for repair that is capable of recognizing a wide variety of lesions that form bulky adducts on DNA bases. Examples of lesions are listed below. DNA lesion Source benzo[a]pyrene diol-epoxide adduct benzo[a]pyrene in tobacco smoke aflatoxin adduct toxin from Aspergillus, which grows on food grains cyclobutane pyrimidine dimer ultraviolet radiation 6-4 photoproduct ultraviolet radiation cisplatin intrastrand crosslink Anticancer drug 9

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11 Diseases of defective NER d) Xeroderma pigmentosum Clinical: Biochemistry: Genetics: dry skin (xeroderma), abnormal pigmentation (pigmentosum) sensitivity to ultraviolet radiation susceptibility to skin cancer mental retardation defective nucleotide excision repair autosomal recessive 7 complementation groups 11

12 Xeroderma pigmentosum (XP) genes Gene Yeast gene Protein function XPA/RPA Rad14/Rpa binds damaged DNA 1000-fold preference XPB Rad25 (SSL2) DNA helicase (3 to 5 ), component of TFIIH XPC/hHR23B Rad4/Rad23 binds damaged DNA XPD Rad3 DNA helicase (5 to 3 ), component of TFIIH XPE/p48? binds damaged DNA with 500,000-fold preference XPF/ERCC1 Rad1/Rad10 DNA endonuclease for 5 side of damage XPG Rad2 DNA endonuclease for 3 side of damage 12

13 e) Other diseases of defective nucleotide excision repair Disease Cancer Clinical features Genes Cockayne's syndrome none skin photosensitivity premature aging cachectic dwarfism mental retardation* trichothiodystrophy (hair-brain syndrome) none skin photosensitivity brittle hair mental retardation CSA, CSB, XPB, XPD, XPG TTD-A, XPB, XPD * Mental retardation in CS and XP are due to different mechanisms. CS shows decreased myelination in the central nervous system, while XP shows neuronal death. 13

14 Transcription Coupled Repair 1. Not all regions of the genome are repaired with equal efficiency. 2. Transcribed DNA is repaired more efficiently than nontranscribed DNA. In fact, the transcribed strand of a gene is repaired more efficiently than the nontranscribed strand of the same gene. 3. Transcription coupled repair (TCR) refers to DNA repair that is somehow activated when RNA polymerase II encounters a DNA lesion. Global genomic repair (GGR) refers to the DNA repair that is independent of transcription. e) Human disease and the repair of UV damage Cells global genomic repair transcription coupled repair UV sensitivity skin cancer wild type human yes yes no no no no yes yes XP groups A, B, D, F, G CS groups A, B yes no yes no XP group C no yes yes yes XP group E no for CPDs yes mild yes wild type rodents no for CPDs yes no yes UV sensitivity always occurs when TCR is defective, but not always when GGR is defective. On the other hand, skin cancer always occurs when GGR is defective, but does not occur when there is an isolated defect in TCR as in Cockayne s syndrome, presumably because the remaining GGR is sufficient to protect against skin cancer. In fact, when rodent cells are transfected with the XPE gene p48, they acquire GGR of CPDs and resistance to UV-induced mutagenesis but show no change in UV sensitivity. 14

15 4) Repair of oxidative damage Mitochondrial respiration generates free radicals and reactive oxygen species: O 2 (superoxide), H 2 O 2 (hydrogen peroxide), and OH (hydroxyl radical) Oxidative damage causes single-strand breaks and damaged bases such as 8-oxoguanine and thymine glycol. Cells contain specific glycosylases to repair the damaged bases. 15

16 Repair of thymine glycols Thymine glycols are removed by the Nth glycosylase (e n donuclease three). Thymine glycols are removed more efficiently from transcribed DNA. CSB, XPG/CS, and XPD/CS mutations lead to defective TCR of thymine glycols. XPG and XPD mutations not associated with CS do not affect TCR of thymine glycols. XPG protein stimulates Nth glycosylase. Hypothesis for the molecular basis of Cockayne's syndrome (1) Defective TCR of oxidative damage blocks RNA polymerase II, leading to dwarfism due to a global transcription defect, and mental retardation due to a specific transcription defect for myelin basic protein. (2) Defective TCR of UV-induced lesions causes UV sensitivity. (3) Intact GGR of UV-induced lesions is enough to prevent skin cancer. 4) Conclusions Cells contain many mechanisms for repairing DNA damage. Several DNA repair genes play other roles in DNA metabolism: TFIIH is involved in transcription initiation and nucleotide excision repair. Double-strand break repair genes are required for generating the immune system. Defects in DNA repair lead to many cancers, including breast, colon, and skin cancer. They also lead to growth and brain abnormalities. Do common polymorphisms in DNA repair genes lead to cancer susceptibility? Will understanding DNA repair help to cure cancer and prevent brain degeneration? References 1. Chu G (1996) Xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy: do the genes explain the diseases? Trends Genet. 12, Chu G (1997) Double-strand break repair. J. Biol. Chem. 272, Croteau D and Bohr V (1997) Repair of oxidative damage to nuclear and mitochondrial DNA in mammalian cells. J. Biol. Chem. 272, Friedberg E, Walker G and Siede W, DNA Repair and Mutagenesis, ASM Press Modrich P (1997) Strand-specific mismatch repair in mammalian cells. J. Biol. Chem. 272, Wood R (1997) Nucleotide excision repair in mammalian cells. J. Biol. Chem. 272,