Semiconservative Replication
DNA Replication
Chapter 11
Wikipedia
Kornberg’s DNA Polymerase I
Requirements for DNA synthesis
DNA Polymerase
Mg++
dNTP
single stranded DNA template
Priming 3’ OH
DNA Polymerase Reaction
Consider how the active site descriminates among the dNTP’s.
Other Enzymatic Activities
Origin of Replication
E. coli DNA Polymerases
I
II
III
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9 mers- TTATTTCCAC
Enzymatic Activities
5’-3’ synthesis
Required Primer
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3’-5’ Exonuclease
(Proofreading Activity)
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Order of Interaction
1. dnaA binds 9mers
2. dnaBC bind dnaA and 13mer -dnaB ATP dependent
helicase
3. ssbp bind unwound DNA stabilizing it
4. Gyrase (topoisomerase) relieves torsional stress
5. Primase (dnaG) synthesizes short primers
6. DNA polymerase III initiates synthesis
5’-3’ Exonuclease
(Replacement Activity)
Pair 13mers GATCTCTTATTAG
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DNA Polymerase III mutation lethal – essential to replication
DNA Polymerase I mutation viable – higher rate of mutation
DNA Polymerase II mutation viable – function unknown
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dnaA
Discontinuous Replication
DNA Polymerase synthesizes
5’-3’ only
http://oregonstate.edu/instruction/bb492/figletters/FigH3.html
Two Replication Forks
Replication Fork
http://oregonstate.edu/instruction/bb492/figletters/FigH2.html
DNA Polymerase Clamp
Improved Processivity
Replication Fork
http://www.wehi.edu.au/education/wehi-tv/dna/replication.html
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Simultaneous Synthesis
Eukaryotic Issues
• Size of Genome
• Ends of Linear Chromosomes
Movie
Size of Eukaryotic Genome
Genome Size
– E coli
– Human
Multiple Origins of Replication
4.6 Mbp genome
3330 Mbp genome (Haploid)
Speed of Polymerase
E. coli
100kb/min
with two replication forks 23 min to copy genome
Humans
1kb/min
with 2 replication forks per chromosome
47.7 days to replicate genome
typical S phase – 2-3 hours
embryonic S phase 3 minutes
Problem with linear chromosomes
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAReplication.html
Eukaryotes can vary origins during development
Telomerase
http://www.steve.gb.com/images/science/telomerase.png
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Telomerase Biology
• Aging and Senescence
• Telomerase and Cancer
http://learn.genetics.utah.edu/features/telomeres/
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