DNA Synthesis
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Transcript of DNA Synthesis
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DNA Synthesis•Eukaryotes
•DNA unwinds at a replication origin •Two replication forks form •Bidirectional synthesis of DNA •Much more DNA; complexed with proteins
•Eukaryotes have much more DNA
•Yeast 250-400 replicons •Mammalian cells up to 25,000
•Solution: Multiple replication origins
•Yeast 4X that of E. coli •Drosophila 100X that of E. coli
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DNA Synthesis in Eukaryotes
Pre-initiation replication complex:ORCCdt1Cdc6MCM
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Enzyme Function
Prokaryotes Eukaryotes
Helicase
Primase
Polymerase
Proofreading
Sliding clamp
Prevent ss closure
DnaB
DnaG
Pol III
Pol III
b sliding clamp
SSBPs
MCM
Primase s.u. of a-polymerase
Pol d and e
Pol d and e
PCNA (Proliferating Cell Nuclear Antigen)
RPA (Replication Protein A)
Comparison of some replication components
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A Problem and a Solution
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Telomere Formation
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A model for t loop formation
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Reproductive cloningBiotechnology/Cloning
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Reproductive cloning
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Therapeutic cloning
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Therapeutic cloning
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First Cloning ExperimentCohen et al., 1973“Recombinant DNA”
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Cloning Vectors: pBR322 plasmid
Cloning into pBR322
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RT-PCR and Cloning