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DNA repair 1 and 2

DNA repair

QuestionAnswer
Deamination Nucleotide bases can undergo spontaneous deamination. Common: Cytosine to uracil. Nitrous acid reacts with bases that contain amino groups to cause deamination.
Deamination examples Adenine oxidatively deaminated to hypoxanthine, methylcytosine to thymine. Guanine to xanthine. Hypoxanthine pairs with cytosine rather than thymine. Xanthine with Cytosine.
Oxidation Oxidizing agents produce reactive oxygen species that can directly damage DNA bases especially guanine. Guanine is oxidized to 8-oxo-guanine.
Alkylating agents Many reactive chemicals can add alkyll groups to nucleotide bases. Methylation can occur. Guanine= O^6 methylguanine which can pair with T.
Base analogs Damage to free bases can lead to their incorporation into DNA after they are converted to triphosphates. ex. 5-bromouracil and 2-aminopurine
Buulky adducts Many polycyclic aromatic hydrocarbon compounds are potent carcinogens. when eliminated they convert to hydrophillic compounds and a side product is epoxides that react with amino groups.
Base hydrolysis-depurination Glycosidic bonds between deoxyribose and purine bases can be hydrolyzed in a spontaneous reaction. Guanosine is most ready. Loss of base leads to apurinic site which have no template info. DNA pol gets block at site.
UV radiation Causes crosslinking of pyrimidine bases, the formation of thymine-thymine dimers. Adjacent thymines are crossed linked and this blocks DNA replication can lead to mutations.
Intercalating agents Acridines can intercalate in DNA, slip in between adjacent base pairs in the DNA double helix. The lead to the insertion or deletion of one or more bases
DNA strand breaks Ionizing radiation cause double strand breaks. Leads to incomplete replication
Mismatch repair in E. Coli MutS and MutL proteins form a complex. MutS recognize damaged DNA. Nick is made.
How does MutS figure out which strand is mismatched? The methylation state of the DNA because E. Coli DNA is methylated and the newly synthesized DNA is unmethylated.
What else does MutL bind to? It binds to MutH which has a latent endonuclease activity, which cleaves the non-methylated strand.
After the unmethylated strand is cleaved what happens? Exo 1, a 3' to 5' exonuclease is recruited and degrades the DNA all the way past the mismatch. A helicase assist in unwinding the DNA. SSB binds to the ssDNA in the gap til Pol III begins to fill gap and ligase seals it.
Mismatch repair in humans MSH (Muts homologs) MutSaplpha is a dimer of 2 MSH proteins, MSH 2 and MSH 6. MutLalpha is a dimer of MLH1 and PMS2 proteins. MutSalpha and MutLalpha form a complex.
What interaction does the human mismatch repair system depend on? Interaction of MutSalpha/MutLalpha complex with PCNA and RFC to initiate repair. Mismatch introduced is likely to be on an Okazaki fragment. PCNA and RFC recognizes 3' primer ends.
How is the removal of DNA accomplished in the human? Exo1, a 5' to 3' exonuclease . A helicase and RPA are involved in the process. Filling the gap is accomplished by Poldelta/PCNA.
Clinical Aspect: Colon Cancer Defects in the MSH2 or MLH1 genes lead to a hereditary disease: Herediatry Nonpolyposis Colon Cancer. Leads to increased risk of other cancers. Intestinal cells are constantly proliferating
Several mutated genes for fully developed cancer examples Tumor supressor genes: Chromosome 5, 18, 17 (p53)and ras oncogene on 12.
Base Excision repair Examples Deaminated, oxidized or methylated bases are repaired by this pathway. Ex. Uracil, hypoxanthine and 8-oxo-guanine
Base Excision repair intiation By a group of DNA glycosylase enzymes. Each recognize specific types of damaged bases. Cleave off damaged bases by hydrolysis of beta-N-glycosidic bond b/w a damaged base and dexyribose. (AP site)
Second step in Base excision repair Cleavage of the phosphodiester bond by an AP endonuclease. It leaves a 3'-hydroxyl and a 5' phosphate end. The end consist of a 5' deoxyribose phosphas.
Filling of gap in E. Coli vs. Human E. Coli: Pol 1 adds severatl nts and removes the 5' deoxyribose phosphate with its 5' to 3' exonuclease activity. Human: DNA glycosylases carry out same thing except there are a larger # of enzymes. Pol beta fills gap.
Pol beta Bifunctional enzyme with a polymerase and a 5' deoxyribose phosphate lyase activity. Inserts a single nt to fill gap. 5' dRP is removed by the 5'dRP lyase activity. DNA ligase seals it
Nucleotide Excision Repair Deals with large or bulky DNA lesions that lead to distortion of the double helix. Repairs pyrimidine dimers formed by UV damage.
Nucleotide Excision Repair process summary Protein complexes recognize the distortions. Recruits helicases that open up DNA. 2 endonucleases cuts are made. Patch of DNA is removed. Gap is filled and ligase seals it.
Nucleotide Excision Repair process in E. Coli UvrA protein recognizes damage, recruits UvrB to open a stretch of about 12 nts. UvrC is recruited and makes cuts. Segment removed by helicase filled by Pol 1.
DNA damage Tolerance Pathways
Base Excision repair Examples Deaminated, oxidized or methylated bases are repaired by this pathway. Ex. Uracil, hypoxanthine and 8-oxo-guanine
Base Excision repair intiation By a group of DNA glycosylase enzymes. Each recognize specific types of damaged bases. Cleave off damaged bases by hydrolysis of beta-N-glycosidic bond b/w a damaged base and dexyribose. (AP site)
Second step in Base excision repair Cleavage of the phosphodiester bond by an AP endonuclease. It leaves a 3'-hydroxyl and a 5' phosphate end. The end consist of a 5' deoxyribose phosphas.
Filling of gap in E. Coli vs. Human E. Coli: Pol 1 adds severatl nts and removes the 5' deoxyribose phosphate with its 5' to 3' exonuclease activity. Human: DNA glycosylases carry out same thing except there are a larger # of enzymes. Pol beta fills gap.
Pol beta Bifunctional enzyme with a polymerase and a 5' deoxyribose phosphate lyase activity. Inserts a single nt to fill gap. 5' dRP is removed by the 5'dRP lyase activity. DNA ligase seals it
Nucleotide Excision Repair Deals with large or bulky DNA lesions that lead to distortion of the double helix. Repairs pyrimidine dimers formed by UV damage.
Nucleotide Excision Repair process summary Protein complexes recognize the distortions. Recruits helicases that open up DNA. 2 endonucleases cuts are made. Patch of DNA is removed. Gap is filled and ligase seals it.
Nucleotide Excision Repair process in E. Coli UvrA protein recognizes damage, recruits UvrB to open a stretch of about 12 nts. UvrC is recruited and makes cuts. Segment removed by helicase filled by Pol 1.
DNA damage Tolerance Pathways
Base Excision repair Examples Deaminated, oxidized or methylated bases are repaired by this pathway. Ex. Uracil, hypoxanthine and 8-oxo-guanine
Base Excision repair intiation By a group of DNA glycosylase enzymes. Each recognize specific types of damaged bases. Cleave off damaged bases by hydrolysis of beta-N-glycosidic bond b/w a damaged base and dexyribose. (AP site)
Second step in Base excision repair Cleavage of the phosphodiester bond by an AP endonuclease. It leaves a 3'-hydroxyl and a 5' phosphate end. The end consist of a 5' deoxyribose phosphas.
Filling of gap in E. Coli vs. Human E. Coli: Pol 1 adds severatl nts and removes the 5' deoxyribose phosphate with its 5' to 3' exonuclease activity. Human: DNA glycosylases carry out same thing except there are a larger # of enzymes. Pol beta fills gap.
Pol beta Bifunctional enzyme with a polymerase and a 5' deoxyribose phosphate lyase activity. Inserts a single nt to fill gap. 5' dRP is removed by the 5'dRP lyase activity. DNA ligase seals it
Nucleotide Excision Repair Deals with large or bulky DNA lesions that lead to distortion of the double helix. Repairs pyrimidine dimers formed by UV damage.
Nucleotide Excision Repair process summary Protein complexes recognize the distortions. Recruits helicases that open up DNA. 2 endonucleases cuts are made. Patch of DNA is removed. Gap is filled and ligase seals it.
Nucleotide Excision Repair process in E. Coli UvrA protein recognizes damage, recruits UvrB to open a stretch of about 12 nts. UvrC is recruited and makes cuts. Segment removed by helicase filled by Pol 1.
Translesion polymerases Are error prone and have low processivity. They bypass different types of damage.
UV damage----Pol eta When Pol delta or epsilon encounters a TT dimer, its progress is blocked and the replication fork is stalled. Pol eta is then brought into play and switches with the replicative polymerase. Pol eta inserts the right base across TT dimer most times
How are double strand breaks generated? By exposure to ionizing radiation, certain chemical mutagens and when replication forks become stalled. Generated in a controlled manner during the process of homologus recomination.
Repair of double strand breaks The information in the undamaged chromosome is used to extend the broken ends to create overlaps that bridge the break.
NHEJ: non homologous end joining Broken ends are rejoined regardless of their sequence
Breast Cancer related to defects in recombinational repair (HR) BRCA2 gene leads to predisposition to breast cancer
Examples of diseases with HR defects Warner's syndrome, Blooms syndrome, Rothmund-Thompson syndrome and Ataxia-telangiectasia
Warner's syndrome Premature graying and thinning of hair, loss of skin elasticity, cataracts, type 2 diabetes mellitus, hypogonadism, osteroporosis and atherosclerosis
Bloom's syndrome Immuno-deficiency, male infertility and female sub-fertility, small body size, sun-induced facial erythema and predisposition to cancer
Rothmund-Thomson syndrome Growth defieciency, photosensitivity with poikilodermatous skin changes, early graying and hair loss, cataracts, increase in cancer incidence, mainly osteogenic sarcomas.
Ataxia-telangiectasia Cerebellar ataxia, immunodeficiency, oculocutaneous telangectasia
Genomic instability Abnormal high rates of genetic change above rate of somatic mutation.