Nucleic Acids Nucleic Acid Function Central Dogma

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 1 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 a -1 sec-1 dNTP polymerized min at 37 °C. 100 10,000 500,000 b b not including Okasaki fragments *In a complex with 10 proteins (26 subunits) of >900 kD. Core is trimer of a, q, e 2 DNA Polymerase Structure DNA/RNA polymerase mechanism 3 11/13/21 DNA/RNA polymerase mechanism Metal Ion Role Proximity Role a b g Asp = O C Asp This is just the chemistry step, there are other more important enzymatic steps… 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 4 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 10–10 mistakes/bp = 0.0003 mistakes/genome – 1 mistake 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 Thumb ddCTP CLOSED OPEN Fingers move Fingers template N H H O N N R N H N Palm N N H O H primer R H N N N R N-term Exo N N N H H O N 2 3 m inor groove O N R 2 3 m inor 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’) 5 Replication/polymerase Fidelity ddCTP closed open Fingers move template primer 2 3 m inor groove Correct 2 3 m inor 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 N H H O N N H N N N N H O R H H N N N 3 2 H m inor groove R N N H O N N Incorrect R O 2 3 m inor groove Incorrect put in E*(open)•dNTP(incorrect) every 10,000 (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’) 6 Replication/polymerase Fidelity DNA Polymerases have 3’ à 5’ Exonuclease Activity Post-insertion site 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 Detected by 3’ à 5’ exonuclease activity (part of DNA polymerase) Phosphodiester bond made Replication Fidelity: Proofreading 7 Replication/polymerase Fidelity Comparison of Polymerases * (a4) 20 5 1000 ?à Processivity a 100 -1 sec-1 dNTP polymerized min at 37 °C. 1,500 500,000 b b not including Okasaki fragments *In a complex with 9-10 proteins (26 subunits) of >1000 kD. Core is trimer of a, q, e DNA Replication Process 8 Pol I Has 5’ à 3’ Exonuclease Activity Nick Translation U U U U 9