DNA Polymerases problems Ch 25:5-9, (5, 7-10) more in depth Study guide: Discussion topics p 308, 11-16, 21-24 The Kornberg enzyme Pol 1 Pol 2 Pol 3 Tom Kornberg The Enzymology of DNA Replication If Watson and Crick were right, then there should be an enzyme that makes DNA copies from a DNA template In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA dependent DNA polymerase - DNA polymerase I, also known as the Kornberg enzyme In 1959, Arthur Kornberg received the Nobel Prize in Medicine for his pioneer work on Pol1 that catalyzes ?the reactions of life? Later work showed that Pol III is the principal replication enzyme in E. coli The three catalytic activities of DNA Polymerase I 5?-3? DNA dependent DNA polymerase activity 3?-5? exonuclease activity or proof reading function 5?-3? exonuclease activity Study guide p. 308, #7, 15-16 The E. coli DNA polymerase I (Mr 103,000) has three catalytic activities: 1. 5?-3? polymerizing activity TACGGTA-OH ATGCCATACGTCCACTTG 5? 3? 3? 5? dTTP, dATP, dGTP, dCTP Pol I cannot start a new chain Pol I is a DNA dependent DNA polymerase Mg++ ss DNA template primer with a 3?-OH TGCAGGTGAAC Base pairing and base stacking interactions between incoming nucleotide and adjacent nucleotide favors polymerization reaction. Polymerase I will not catalyze the formation of the phosphoester bond unless bases are properly paired - ensures fidelity of replication. Makes a bond and breaks a bond DG = 0 Accuracy is about 1 mistake in every 104-105 bp. Mg2+ coordinates the P ? ? ? ? See animation on course website reversible (dNMP)n + dNTP (dNMP)n+1 + PPi Net DG=0 Polymerization is a thermodynamically favorable reaction ? ? ? The correct base pairs Have very similar geometry Fidelity of DNA synthesis is dependent on the fidelity of Watson-Crick base pairing 2. 3?-5? exonuclease activity (editing or proof reading function) Exonuclease activity is enhanced by mismatch or lack of dNTPs Proof reading improves accuracy by 102-103 folds. Repair mechanisms improves accuracy by another 102-103 folds. Genetic analysis shows that the overall error rate for the duplication of genetic information is 1/109-1/1010 bp. Pol I can resynthesize a stretch of DNA by chain elongation at the nick and removing nucleotides ahead to clear the way for polymerization. 3. 5?-3? exonuclease activity This activity is enhanced by concomitant DNA synthesis in a coordinated mechanism for repair synthesis known as nick translation. The Klenow fragment of DNA polymerase I with bound DNA Hans Klenow used either subtilisin or trypsin to cleave between residues 323 and 324, separating 5'-exonuclease (on 1-323) and the other two activities (324-928, the so-called "Klenow fragment?) Figure 25.8 Tom Steitz The Klenow fragment lacks the 5?-3? exonuclease activity Used for filling in gaps. No nick translation activity. The larger the enzyme, the harder it is to crystallize. Can be cleaved into two portions? Very useful because it lacks the 5 to 3 exonuclease activity. Fills in a gap?it is a partial enzyme Primer (14 nucleotides) Template (12 nucleotides) 5? 3? A Pol I mutant with only 1% of the 5?-3? polymerase activity is viable. ?Is Pol I ?the? replication enzyme ?? - asked John Cairns Search for the real polymerase ! Enters Tom Kornberg and Malcom Gefter Pol I accounts for 90% of the DNA polymerase activity of E. coli Molecules/cell 400 ND ~40 dimer - A dimer DNA Polymerase III holoenzyme Clamp loader complex 5? 5? 3? 3? 3? 5? 3? 5? 5? 3? Two b subunits of Pol III form a clamp around the DNA duplex to prevent dissociation of Pol III from the DNA duplex. The b subunits are ATPases required for optimal processivity. fig. 25.10
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