DNA REPLICATION IN PROKARYOTES Origin of Replication (oriC) o Controls replication of a replicon o Prokaryotes have 1, eukaryotes have 2 o One 13-bp sequence present at 3 repeats rich in A:T, facilitating formation of replication bubble o 9-bp sequence repeated 4 times acting as binding site for protein needed in formation of replication bubble o oriC unwinds easier than other DNA because the 9-bp and 13-bp repeats create a good unwinding site Biodirectional Replication o 2 replication forks (Y shaped structures) move in opposite directions around circular chromosome DNA ligase converts H-bonded circular molecule to closed circular molecule o Seals strand breaks in DNA helices o Required in all organisms for DNA replication, repair and recombination Denaturation: exposure to high temperature or pH that causes 2 strands in double-helix to separate One strand extended 5’ 3’ and other strand extended 3’ 5’ DNA polymerase catalyzes replication of DNA, but only carried out in 5’ 3’ Leading strand (5’ 3’) is continuous , but lagging strand (3’ 5’), since can’t be catalyzed by DNA polymerase, grows by discontinuous synthesis of short fragments (5’ 3’) called okazaki fragments Okazaki fragments are then linked together by DNA ligase : catalyze covalent closure of nicks DNA ligase and DNA polymerase together can fill in and seal any present gaps Initiation Begins at oriC with formation of replication bubble Each new DNA chain is initiated by a short RNA primer synthesized by DNA primase DNA polymerase I : o Removes RNA primers and replaces with DNA chains o 5’ 3’ polymerase o 5’ 3’ exonuclease activity removes RNA primer and replaces with DNA chain o proofreading: 3’ 5’ exonuclease activity splits off nucleotides from 3’ termini DNA polymerase III : o Main replicative polymerase, highly processive o Catalyzes semiconservative replication of the chromosome o Catalyzes addition of DNA to RNA primers o Can terminate Okazaki fragments
o 5’ 3’ polymerase activity o no 5’ 3’ exonuclease activity o proofreading has 3’ 5’ exonuclease activity DNA replication requires two double-helix strands to separate DNA helicases unwind DNA molecule SSB protein keeps strands in extended form to prevent them from forming hairpin structures DNA topoisomerase: enzymes that provide required axes of rotation during replication of circular DNA o Topoisomerase I: produce temporary single-strand nicks in DNA which provide axis of rotation, also remove supercoils one at a time o Topoisomerase II: enzymes produce transient double-strand breaks in DNA, also add or remove supercoils 2 at a time Proof reading: scanning termini of nascent DNA chains for errors and correcting them carried out by 3’ 5’ exonuclease activity of DNA polymerases o When a template primer DNA has mismatched base pair at 3’, the 3’ 5’ exonuclease activity clips off mismatched base. When appropriately
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- Fall '13
- DNA, RNA molecules, rna polymerase