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DNA Replication
Part 2
Enzymology
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Figure 11.10
The Polymerization Reaction
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Main E. coli DNA Polymerases
• DNA polymerase I– Single subunit enzyme – Degrades RNA primers and fills gaps– DNA repair– 5'3' polymerase activity– 3’5’ exonuclease activity – proofreading function– 5’3’ exonuclease activity – primer removal & repair function
• DNA polymerase III– Main DNA polymerase for replication of genome and plasmids– Multiple subunit holoenzyme – different subunits for pol and exo
activities– 5'3' polymerase activity– 3’5’ exonuclease activity – proofreading function
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Eukaryotic DNA Polymerases
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Catalytic domain of E. coli DNA Polymerase I
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holoenzyme
Pol III*
E. coli DNA Pol III Holoenzyme
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Sliding Clamps
E. Coli dimer clamp Human trimer clamp
Subunit PCNA
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Enzymology of DNA Synthesis
• DNA-dependent DNA Polymerases– require a DNA template
• Require a free 3'-OH– a primer
• NTP added to 3'-OH of growing polynucleotide chain– 5' 3' synthesis
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Mechanics of DNA Synthesis
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Architecture of the Replisome
535
3
35
5
HelicaseTopoisomerase
ssDNA-bindingproteins
DNApolymerase RNA
primer
Leading strand
Lagging strand RNAprimer
Primase
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DNA Pol I Finishes the Job
• DNA Pol I has 5'3' exonuclease function that destroys RNA primer
• As RNA is removed, DNA is replaced by the 5'3' polymerase function
• DNA ligase seals the gap by joining the 3'OH to the 5'PO4
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Termination of Replication: Circular Chromosomes
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Figure 11.24
What happens at the end of a linear chromosome?
• The need for a primer poses a problem at the 3’ end of linear chromosomes
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• Linear eukaryotic chromosomes have telomeres at both ends
• The term telomere refers to the complex of telomeric DNA sequences and bound proteins
Telomeres and DNA Replication
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Figure 11.23
• Telomeric sequences consist of hexanucleotide or heptanucleotide repeats
• The very end of telomeres are a 12-16 nucleotides 3’ overhang formed by removal of the RNA primer after DNA synthesis
Telomere Sequence & Structure
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Replication of Telomere Sequences
• In mammalian somatic cells, 100-1000 telomere repeats are lost with each round of replication
• Telomere sequence must be maintained in gamete precursors– Done by telomerase
• Telomerase– utilized a RNA template to extent the 3’ end of one strand
at the telomere
– telomerase has reverse transcriptase enzyme activity
– The RNA is complementary to the DNA sequence found in the telomeric repeat
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Figure 11.25
Step 1 = Binding
Step 3 = Translocation
The complementarystrand is made by primase, DNA polymerase and ligase
RNA primer
Step 2 = PolymerizationReverse transcriptase
Telomerase & Telomere Extension
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Telomeres Protect Ends of Chromosomes