Nucleic Acids DNA Replication

Nucleic Acids A. The 4 S’s of Nucleotides & NA B. Structure of the Information C. Recombinant DNA: Biochemical Basis of Biotechnology 1. Restriction enzymes, DNA ligase 2. Vectors and Inserts to make recombinant DNA (rDNA) 3. Transformation of hosts 4. Selection of transformants 5. Expression 6. Site-directed mutagenesis D. Replication 1. Polymerases 2. Fidelity a. b. c. d. Polymerase recognition Exonuclease Mis-match repair Post-replication repair i. Direct reversal ii. Base excision iii.Nucleotide excision 3. Sequence determination 4. PCR E. Transcription 1. RNA polymerase 2. fidelity F. Translation 1. Genetic code 2. tRNA 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 Replication Nucleic acid function: Central Dogma DNA Replication Addition at 3’ end DNA Polymerases Require 3 things: Pyrophosphatase dNTP 2 Pi DG°’ ≈ –3 kcal/mole DG°’ ≈ –6 kcal/mole DG°’ ≈ –9 kcal/mole Comparison of Polymerases * (a4) 20 5 1000 ?à ?à Processivity adNTP -1 at 37 °C. sec-1 polymerized min 100 10,000 bnot 500,000 including Okasaki fragments *In a complex with 10 proteins (26 subunits) of >900 kD. Core is trimer of a, q, e DNA Polymerase Structure b 11/16/20 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 Replication/polymerase Fidelity Geometry of Base Pairing Accounts for High Fidelity • Errors in replication for E. coli: 1/109 – 1/1010 bp – 3x106 bp/genome x 1/10–10 mistakes/bp = 0.0003 mistakes/genome – 1 per 1,000–10,000 replications • DNA polymerase active site excludes base pairs with incorrect geometry – At BOTH the insertion site and the post insertion site – BUT, DNA polymerases still insert wrong base 1/10,000 times. – Repair mechanisms fix these errors. D D D D Base-Pair Geometry Replication/polymerase Fidelity ddCTP closed open template N H H O N N R N H N N N H O H primer Fingers move N R H N N N R N N H H O N 2 3 minor groove N R O 2 3 minor groove E*(open)•dNTP(incorrect) (correct) à E***(translocation)•PP à E + PP dNTP(incorrect) E + dNTP à E*(open)•dNTP à E**(closed)•dNTP i i à binding slow chemistry Movement (5’à3’) Replication/polymerase Fidelity ddCTP closed open Fingers move template primer 2 3 minor groove Correct 2 3 minor groove E + dNTP à E*(open)•dNTP à E**(closed)•dNTP(correct) à E***(translocation)•PPi à E + PPi binding slow chemistry Movement (5’à3’) Replication/polymerase Fidelity ddCTP closed open Fingers move template N primerR H H O N N N H N N N N H O R H H N N R N 3 2 H minor groove N N H O This image cannot currently be displayed. N N Incorrect R O 2 3 minor groove E*(open)•dNTP(incorrect) (correct) à E***(translocation)•PP à E + PP dNTP(incorrect) E + dNTP à E*(open)•dNTP à E**(closed)•dNTP i i à binding slow chemistry Movement (5’à3’) Replication/polymerase Fidelity DNA Polymerases have 3’ à 5’ Exonuclease Activity Replication/polymerase Fidelity DNA Polymerases have 3’ à 5’ Exonuclease Activity The 3’ à 5’ exonuclease domain wrong base Phosphodiester bond made correct base Phosphodiester bond hydrolyzed Phosphodiester bond made Detected by 3’ à 5’ exonuclease activity (part of DNA polymerase) Replication Fidelity: Proofreading Replication/polymerase Fidelity Comparison of Polymerases * (a4) 20 5 1000 ?à Processivity adNTP -1 at 37 °C. sec-1 polymerized min 100 1,500 bnot 500,000 including Okasaki fragments *In a complex with 9-10 proteins (26 subunits) of >1000 kD. Core is trimer of a, q, e b DNA Replication Process Pol I Has 5’ à 3’ Exonuclease Activity Nick Translation U U U Replication/polymerase Fidelity 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 Base-excision repair Nucleotide excision repair 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 separate the two strands and the exonuclease completely removes the defected strand. The gap is then filled by DNA polymerase and DNA ligase. 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 separate 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) 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 can be reversed by “alkyltransferase” (alkB) AlkB demethylates 1-methyladenine and 3-methylcytosine. DNA Repair 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 DNA Repair DNA Repair (II): Base excision Repair abasic endo-nuclease mechanism 1 Base is changed in a small way 2 3 5’ • 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. 3’ 5’ 3’ 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 DNA Repair (II): Base excision Repair STEP 1 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 What if a glycosidase does not exist and/or the lesion is too big? 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 (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) 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) DNA Sequencing Visualize the DNA Sequence (I) 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 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