080818.PMCB.lecture.5

080818.PMCB.lecture.5 - lant Molecular and Cellular Biology...

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Unformatted text preview: lant Molecular and Cellular Biology Lecture 5: DNA Replicase Structure & Function Gary Peter Learning Objectives 1. List and explain the mechanisms by which eukaryote DNA is replicated 2. Describe and explain the structure and functions of the enzymes and their subunits that replicate DNA in eukaryotes Replisome Close Association of Proteins into a Replisome at the Fork z DNA polymerase III holocomplex z Primosome z DNA helicase and DNA primase located at the center of the fork where the two strands of the helix are unwinding bound to DNA pol III Model for the Spatial Organization of the the Replisome 2003 Molecular Microbiology , 49 , 1157–1165 DNA Polymerase III - Holoenzyme z A holoenzyme is the fully functional form of an enzyme which contains all of the necessary subunits to be fully active z DNA Polymerase Holoenzyme z Core enzyme z The sliding clamp z Clamp loading complex Comparison of DNA polymerases I and III DNA polymerase III DNA polymerase I Structure DNA Pol III holoenzyme is an asymmetric dimer; i. e., two cores with other accessory subunits. It can thus move with the fork and make both leading and lagging strands. DNA Pol I is a monomeric protein with three active sites. It is distributive, so having 5'-to-3' exonuclease and polymerase on the same molecule for removing RNA primers is effective and efficient. Activities Polymerization and 3'-to-5' exonuclease, but on different subunits. This is the replicative polymerase in the cell. Can only isolate conditional-lethal dnaE mutants. Synthesizes both leading and lagging strands. No 5' to 3' exonuclease activity. Polymerization, 3'-to-5' exonuclease, and 5'-to-3' exonuclease (mutants lacking this essential activity are not viable). Primary function is to remove RNA primers on the lagging strand, and fill-in the resulting gaps. Vmax (nuc./sec) 250-1,000 nucleotides/second. This is as fast as the rate of replication measured in Cairns' experiments. Only this polymerase is fast enough to be the main replicative enzyme. 20 nucleotides/second. This is NOT fast enough to be the main replicative enzyme, but is capable of "filling in" DNA to replace the short (about 10 nucleotides) RNA primers on Okazaki fragments. Processivity Highly processive. The beta subunit is a sliding clamp. The holoenzyme remains associated with the fork until replication terminates. Distributive. Pol I does NOT remain associated with the lagging strand, but disassociates after each RNA primer is removed. Molecules/cell 10-20 molecules/cell. In rapidly growing cells, there are 6 forks. If one processive holoenzyme (two cores) is at each fork, then only 12 core polymerases are needed for replication. About 400 molecules/cell. It is distributive, so the higher concentration means that it can reassociate with the lagging strand easily....
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080818.PMCB.lecture.5 - lant Molecular and Cellular Biology...

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