Nucleic Acids Replication Polymerase Fidelity Lecture Note

Replication/polymerase Fidelity Mechanism of Mismatch Repair Replication/polymerase Fidelity Mechanism of Mismatch Repair In E. coli a mismatch is recognized by MutS, which then recruits MutL. In an ATP dependent manner, the complex translocates along the double stranded DNA to generate a loop. On encountering a hemi-methylated GATC sequence, it recruits MutH (an endonuclease) and MutH cuts the unmethylated DNA. A helicase (UvrD) then separates the two strands and the exonuclease completely removes the defected strand. The gap is then filled by DNA polymerase and DNA ligase. 2 Replication/polymerase Fidelity Mechanism of Mismatch Repair In E. coli a mismatch is recognized by MutS, which then recruits MutL. In an ATP dependent manner, the complex translocates along the double stranded DNA to generate a loop. On encountering a hemi-methylated GATC sequence, it recruits MutH (an endonuclease) and MutH cuts the unmethylated DNA. A helicase (UvrD) then separates the two strands and the exonuclease completely removes the defected strand. The gap is then filled by DNA polymerase and DNA ligase. DNA Repair Polymerases make mistakes in synthesis (polymerase 5’à3’ activity) = 1/10,000 DNA polymerases have proofreading ability (3’à5’ exonuclease activity) = 1/10,000 Overall for Replication = 1/1010 After replication fork, any mistakes are corrected by mismatch repair mechanism. This misses = 1/100 3x109 bp/haploid genome x 1/1010 mistakes/bp = 0.3 mistakes/haploid genome Plus DNA can be damaged in living cells. These damages are repaired by other mechanisms called Post-replication DNA Repair: • • • Direct reversal of modification (I) Base-excision repair (II) Nucleotide excision repair (III) 3 DNA Repair DNA Repair (I): Direct Reversal of Modification UV-crosslinking: pyrimidine dimers DNA photolyase DNA Repair Repair of Pyrimidine Dimers with Photolyase Another example of direct reversal: O6-alkylguanine modification, and others, can be reversed by “alkyltransferase” (ada) AlkB demethylates 1-methyladenine and 3-methylcytosine. 4 Direct Repair of Alkylated Bases by AlkB RECALL: and other bases Oxidations/Alkylations: • Methylation of G at O6 can base-pair with C or T, which causes G à A mutations 3-methyladenine 5 DNA Repair DNA Repair (II): Base excision Repair abasic endo-nuclease mechanism 1 • Hydrolysis of phosphodiester 5’-side of abasic site • Asp acts as a general base, abstracts proton from water to make it a better nucleophile. • Pentavalent phosphate transition state formed from attack of water • Mg2+ stabilizes the charge that forms on the 3’ oxygen when the phosphate bond is cleaved. Base is changed in a small way 2 3 5’ 3’ 3’ 5’ 5’ 4 5 1. 2. 3. 4. 5. Glycosylase removes damaged base by cleavage of glycosidic bond Abasic-site endonuclease cleaves phosphodiester bond Exonuclease removes abasic deoxyribose Polymerase required to add new nucleotide Ligase condenses phosphodiester bond DNA Repair STEP 1 DNA Repair (II): Base excision Repair Depends on there being a specific Glycosylase [Glycosylases exist for 8-Oxo-guaninie (8-hydroxyG), uracil, hypoxanthine, 3-methyladenine] Oxo-guanine glycosylase Bends DNA helix and extrudes damaged base into enzyme active site Uracil DNA glycosylase Flips out base and fills hole with Arg Recall its in syn What if a glycosidase does not exist and/or the lesion is too big? 6 DNA Repair DNA Repair (III): Nucleotide Excision Repair (NER) Most common and versatile mechanism of repair Bulky alkylation or other distortion of the double helix UvrABC endonuclease (ATP-driven) (A2B2 tetramer bends DNA at damage) P UvrD (helicase) (UvrC is the endonuclease) DNA Repair NER in Bacteria and Humans Lesions include: pyrimidine dimers (formed from UV light) 6,4-photoproducts (from UV light) benzo[a]pyreneguanine (from cigarette smoke) 7 DNA Replication • DNA Replication – Polymerases • Activities • Structure • Mechanism • Replication/polymerase fidelity – Exonuclease Activities – Mismatch repair – Post-replication repair • Reversal • Base Excision repair • Nucleotide Excision repair • DNA Sequencing • PCR DNA Sequence Determination (Sanger) Frederick Sanger (1918-2013) 8 DNA Sequencing Visualize the DNA Sequence (I) 9 Chain-Terminator Sequencing Method DNA Replication • DNA Replication – Polymerases • Activities • Structure • Mechanism • Replication/polymerase fidelity – Exonuclease Activities – Mismatch repair – Post-replication repair • Reversal • Base Excision repair • Nucleotide Excision repair • DNA Sequencing • PCR 10 Molecular Cloning: Polymerase Chain Reaction Besides using host to MAKE MORE copies of DNA sequences, they can be made by the polymerase chain reaction (PCR) technique (in vitro). PCR is a cyclical process: • DNA fragments are denatured by heating g single-stranded DNA • Primers (one complimentary to each strand), plus dNTPs, and DNA polymerase are added g annealing and extension g new double-stranded DNA • Repeat An initial problem with PCR was its temperature requirements. The key insight was the use of an enzyme that could withstand the heat needed to denature the DNA g DNA polymerase from Thermus aquaticus, Taq Polymerase (insight of Kerri Mullis). The amplified DNA can then be inserted into plasmids to create recombinant DNA and cloned in host cells. Synthetic DNA can be manipulated to create specific mutations in order to study the consequences of the mutation: Sitedirected Mutagenesis. Process Diagram: Polymerase Chain Reaction (PCR) Molecular Cloning: Polymerase Chain Reaction 11