DNA Repair Mechanisms PDF

DNA REPAIR DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases. Note: If the damage is not repaired, a permanent change (mutation) is introduced that can result in any of a number of deleterious effects, including loss of cont...

DNA REPAIR DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases. Note: If the damage is not repaired, a permanent change (mutation) is introduced that can result in any of a number of deleterious effects, including loss of control over the proliferation of the mutated cell, leading to cancer. Physical mutagens High-energy ionizing radiation: X-rays and g-rays ® strand breaks and base/sugar destruction Nonionizing radiation : UV light® pyrimidine dimers Chemical mutagens Base analogs: direct mutagenesis Nitrous acid: deaminates C to produce U Alkylating agents Lesions-indirect mutagenesis Intercalating agents Most of the repair systems involve: 1. Recognition of the damage (lesion) on the DNA 2. Removal or excision of the damage, 3. Replacement or filling the gap left by excision using the sister strand as a template for DNA synthesis, 4. And ligation. These excision repair systems remove one to tens of nucleotides. Mismatch repair Which strand is new and which is the parent? The mutation is in the new strand! A-CH3 marks the parental strand! Sometimes replication errors escape the proofreading function during DNA synthesis, causing a mismatch of one to several bases. ØIn E. coli, mismatch repair (MMR) is mediated by a group of proteins known as the Mut proteins Homologous proteins are present in humans. 1. Identification of the mismatched strand: the Mut proteins that identify the mispaired nucleotide(s) 2. Discrimination is based on the degree of methylation. GATC sequences, which are found approximately once every thousand nucleotides, are methylated on the adenine (A) residue. 3. This methylation is not done immediately after synthesis,).The newly synthesized DNA is hemimethylated (that is, the parental strand is methylated, but the daughter strand is not The daughter strand that gets repaired. Mismatch repair -- MSH proteins -- eukaryotes Repair of damaged DNA: 1. Strand containing the mismatch is identified, 2. An endonuclease nicks the strand, 3. And the mismatched nucleotide(s) is/are removed by an exonuclease. 4. Additional nucleotides at the 5 - and 3 -ends of the mismatch are also removed. 5. The gap is filled, using the sister strand as a template, by a DNA polymerase, typically DNA pol I 6. The 3 –OH of the newly synthesized DNA is joined to the 5 -P of the remaining stretch of the original DNA strand by DNA ligase  Mutation to the proteins involved in mismatch repair in humans Is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Ø With HNPCC, there is an increased risk for developing colon cancer (as well as other cancers); however, only about 5% of all colon cancer is the result of mutations in mismatch repair.  A DNA glycosylase initiates base excision repair Examples of bases cleaved by DNA glycosylases: Ø nitrous acid, deaminates cytosine, cytosine, deamination (the loss of its amino group) uracil, adenine (to hypoxanthine), and guanine (to xanthine). Lesions involving base alterations or loss can be corrected by BER Damaged base deamination A* C A A* T C C G Base excision repair ►DNA glycosylases recognize the damage to a single base. ►A glycosylase cleaves the N-glycosidic bond that joins the damaged base to deoxyribose. ► The sugar–phosphate backbone of the DNA now lacks a base at this site (known as an apurinic or apyrimidinic site, (an AP site) ►Then an AP endonuclease cleaves the sugar– phosphate strand. ► the same types of enzymes involved in other types of repair mechanisms restore this region to normal CàU (spontaneous deamination) AP site (apurinic or Uracil repair apyrimidinic)  Exposure of a cell to ultrtaviolet (UV) light can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. thymine dimers prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER) as A related pathway is present in humans UV: covalent cross-link intrastrand x-link: Can’t fit in double helix DNA replication interstrand x-link:  Recognition and excision of dimers by UV- specific endonuclease: 1. A UV-specific endonuclease recognizes the dimer and cleaves the damaged strand on both the 5 -side and 3 -side of the dimer. 2. A short oligonucleotide containing the dimer is released, leaving a gap in the DNA strand 3. that formerly contained the dimer. This gap is filled in using a DNA polymerase and DNA ligase. Nucleotide excision repair 2. UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to unfiltered sunlight. Ø In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers Ø XP can be caused by defects in any of the several genes that code for the XP proteins required for nucleotide excision repair of UV damage in humans. Xeroderma Pigmentosum 23 Repair of double-strand breaks (dsDNA breaks) Ionizing radiation or oxidative free radicals can cause it or during gene rearrangements. cannot be corrected by the previously described strategy of excising the damage on one strand and using the remaining strand as a template for replacing the missing nucleotide(s). Instead, they are repaired by one of two systems: Double-strand break repair NO TEMPLATE FOR REPAIR!! 1. Nonhomologous end-joining (NHEJ), Ø in which a group of proteins mediates the recognition, processing, and ligation of the ends of two DNA fragments. Ø However, some DNA is lost in the process àerror prone and mutagenic. ØDefects in this repair system are associated with a predisposition to cancer and immunodeficiency syndromes. Double-strand break repair NO TEMPLATE FOR REPAIR!! 2. homologous recombination (HR) Ø Uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. Ø This system is much less error prone than NHEJ because any DNA that was lost is replaced using homologous DNA as a template. e.g Mutations to the proteins, BRCA1 or 2 (breast cancer 1 or 2) risk for developing breast cancer.  Double-strand break repair Two basic mechanisms: End-joining and Recombination The end-joining pathway of ds break repair is mutagenic, because it removes several base pairs at the break site. Mediated by Ku proteins. mechanism of DNA double-strand break repair is multistep 1. proteins (protein complexes) that: identify DSBs in genomic DNA (sensors) 2. transduce and amplify the recognized DNA damage (transducers and mediators), 3. molecules that dictate the ultimate outcomes of the DNA damage response (effectors). DNA repair defects cause disease Thank you