DNA Repair Mechanisms: Chapter 25 PDF

DNA Repair Chapter 25 Dr. Suheir Ereqat 2024/2025  Sources of damage endogenous damage: such as attack by ROS and replication errors exogenous damage caused by external agents: UV light, x-rays and gamma rays plant toxins human-made mutagenic chemicals, DNA intercalating agents, Alk...

DNA Repair Chapter 25 Dr. Suheir Ereqat 2024/2025  Sources of damage endogenous damage: such as attack by ROS and replication errors exogenous damage caused by external agents: UV light, x-rays and gamma rays plant toxins human-made mutagenic chemicals, DNA intercalating agents, Alkylating agents viruses Dr. Suheir Ereqat 2024/2025 Mutation: A permanent change in the nucleotide sequence. Categorized by the A) nature of bases - Substitution mutation: transition, transversion - Insertion or deletion mutation: B) effect on coding sequence - Silent mutation do not alter the amino acid encoded - Missense mutation: change amino acid encoded - Nonsense mutation: stop codon Dr. Suheir Ereqat 2024/2025 Base substitution: Transitions Vs transversions Dr. Suheir Ereqat 2024/2025  Mutations and Cancer Ames Test for Carcinogens, based Salmonella typhimurium having a mutation Cant synthesize histidine on their mutagenicity: Measure the potential of a chemical to induce mutations in bacteria(may act as a carcinogen) histidine-free medium Filter disc: mutagen increases the rate of back-mutation and hence the number of colonies Dr. Suheir Ereqat 2024/2025  Ames test for carcinogens, based on their mutagenicity. A strain of Salmonella typhimurium having a mutation that inactivates an enzyme of the histidine biosynthetic pathway is plated on a histidine-free medium, But few cells grow?? -The few small colonies of S. typhimurium that do grow on a histidine-free medium plate (a). carry spontaneous back-mutations that permit the histidine biosynthetic pathway to operate. -Three identical nutrient plates (b), (c), and (d) have been inoculated with an equal number of cells. Each plate then receives a disk of filter paper containing progressively lower concentrations of a mutagen. The mutagen greatly increases the rate of back-mutation and hence the number of colonies. The clear areas around the filter paper indicate where the concentration of mutagen is so high that it is lethal to the cells. As the mutagen diffuses away from the filter paper, it is diluted to sublethal concentrations that promote back-mutation. Mutagens can be compared on the basis of their effect on mutation rate. Dr. Suheir Ereqat 2024/2025  Fidelity of DNA replication The fidelity of DNA replication is maintained by: (1) base selection by the polymerase, (2) a 3>5 proofreading exonuclease activity that is part of most DNA polymerases, (3) specific repair systems for mismatches left behind after replication Dr. Suheir Ereqat 2024/2025  Deamination of C= Uracil Deamination of A=hypoxanthine Deamination of G=Xanthine Dr. Suheir Ereqat 2024/2025 1) Methyl- directed Mismatch Repair: tagging by Dam methylase at(5)GATC Palindromic sequence A palindromic: sequence is a nucleic acid sequence on double stranded DNA where in reading 5' to 3' forward on one strand matches the sequence reading 5' to 3' on the complementary strand with which it forms a double helix Dr. Suheir Ereqat 2024/2025 Methyl- directed mismatch repair: lesion The MutL protein forms a complex with MutS at the mismatch. MutH protein binds to MutL and to GATC sequences encountered by the MutL-MutS complex, DNA on both sides of the mismatch is threaded through the MutL-MutS complex= DNA loop Dr. Suheir Ereqat 2024/2025 Mismatch at 3’ side of the cleavage site Mismatch at 5’ side of the cleavage site both directions 5>3 direction. Degradation:5>3 direction Degradation:3>5 direction Dr. Suheir Ereqat 2024/2025 Eukaryotes: - Similar to MutS and MutL. - MutS homologus for eukaryotes from yeast to humans. MSH2 (MutS homolog 2) MSH3, MSH6. Homologs of MutL, predominantly a heterodimer of MLH1 (MutL homolog 1) and PMS1 (post-meiotic segregation), bind to and stabilize the MSH complexes. - Mutated in CANCER= mutation rate Dr. Suheir Ereqat 2024/2025 Eukaryotes: Many details of the subsequent events in eukaryotic mismatch repair remain to be worked out. In particular, we do not know the mechanism by which newly synthesized DNA strands are identified, although research has revealed that this strand identification does not involve GATC sequences. Dr. Suheir Ereqat 2024/2025 2) Base- Excision Repair:recognize and repair damage caused by environmental agents. cleaving the N-glycosyl bond abasic site (AP site) Example: deamination of cytosine=Uracil removed by Uracil DNA glycosylases, human=4 UNG, initiates repair synthesis from the free 3 OH at the nick, removing (with its 5>3 exonuclease activity) and replacing aportion of the damaged strand. Dr. Suheir Ereqat 2024/2025  recognize and remove bulky lesions and pyrimidine 3) Nucleotide Excision Repair: dimers. bulky lesion Three subunits: UvrA,B,C dual incision two specific endonucleolytic cleavages Gap!! Nick!! Dr. Suheir Ereqat 2024/2025 This pathway the primary route for many lesion types : - pyrimidine dimers (T dimer). - base adducts: benzo pyrene-guanine (formed in DNA by cigarette smoke). DNA adduct Dr. Suheir Ereqat 2024/2025 Xeroderma pigmentosum (XP): rare inherited disease ( pigmented lesions on skin + skin cancer+ also have neurological abnormalities) due to mutations(XPA-XPG) in Nucleotide Excision Repair system (the sole repair pathway for pyrimidine dimers in humans). Dr. Suheir Ereqat 2024/2025 HNPCC (hereditary non-polyposis colorectal cancer) a genetic disease of autosomal dominant inheritance = defect miss match repair It can present with rectal bleeding, stomach pain and cancer-related symptoms like unexplained weight loss and fatigue. The most prevalent are defects in the hMLH1 (human MutL homolog 1) and hMSH2 (human MutS homolog 2) Dr. Suheir Ereqat 2024/2025 DNA damage cause mutations: O6-methylguanine forms tends to pair with thymine rather than cytosine during replication, and therefore causes G-C to A-T mutations Dr. Suheir Ereqat 2024/2025 4) Direct Repair: Repair with no excision / removal of a base or a nucleotide. A) Photoreactivation: Pyrimidine dimers result from a UV-induced reaction, and photolyases use energy derived from absorbed light to reverse the damage Dr. Suheir Ereqat 2024/2025 T C dimer Dr. Suheir Ereqat 2024/2025  UV light strikes one of the adjacent thymines, creating a thymine dimer. DNA photolyases recognize the “kink” in coded for by phr genes the DNA, and bind to the site When excited by blue light (350-500 nm wavelength), the photolyases change conformation, breaking apart the dimer. Dr. Suheir Ereqat 2024/2025 Dr. Suheir Ereqat 2024/2025 B)-repair of nucleotides with alkylation damage transfer of the methyl group to its own Cys residues. Dr. Suheir Ereqat 2024/2025  c- Direct Repair: oxidative demethylation of alkylated nucleotides by the AlkB protein, Demethylation by AlkB is accompanied with release of CO2, succinate and formaldehyde The AlkB enzyme couples oxidative decarboxylation of -ketoglutarate to the hydroxylation of the methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde. Dr. Suheir Ereqat 2024/2025 There are nine human homologs of AlkB. ALKBH1, ALKBH2, ALKBH3, ALKBH4, ALKBH5, ALKBH6, ALKBH7, ALKBH8, FTO Dr. Suheir Ereqat 2024/2025  The Interaction of Replication Forks with DNA Damage Repair must come from homologous chromosome SOS response: Dr. Suheir Ereqat 2024/2025  cell cycle is arrested repressor activator activator Dr. Suheir Ereqat 2024/2025  SOS Repair Dr. Suheir Ereqat 2024/2025 1-Recombinational DNA repair. 2-Error prone translesion DNA synthesis (TLS). UmuD’ complex with UmuC DNA pol V replicate many lesions that normally would block replication. Pol IV, induced under SOS response which is also highly error-prone.. Proper base pairing is nearly impossible inaccurate repair + high mutation rate. SOS activated UmuD’ +UmuC only when all replication forks blocked { result of extensive DNA damage}. The bacterial DNA polymerases IV and V are part of a family of TLS polymerases found in all organisms. These enzymes lack a proofreading exonuclease activity thus have low fidelity. Other polymerases in eukaryotes: DNA polymerase eta, iota. Dr. Suheir Ereqat 2024/2025 Dr. Suheir Ereqat 2024/2025