Lecture 10 - Lecture10...

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Lecture 10 Fig 18.3, 18.5 DNA polymerase various nuclease activities DNA polymerase 1 has a unique 5’-3’ exonuclease activity it can be combined with DNA synthesis to perform nick translation Nick - cut between phosphodiester bonds Gap - has a missing nucleotide on one of the two strands. cold DNA - DNA that is not radioactively labeled In nick translation you add two enzymes, one is called DNAase 1 and DNA polymerase 1 DNAase 1 - makes singles strands mix in a non specific manner DNA polymerase 1 is one of the only polymerases that the 5’-3’ exonuclease subunit is actually part of the same polypeptide that has the catalytic function as well. it is the repair enzymes, only used to fix errors in DNA, and removes the RNA primer DNA polymerase 3 (replicase) uses he 5’-3’ exonuclease of DNA polymerase 1 to get rid of the RNA primers Fig 18.10 Action of helicases replication requires a helicase to separate the strands of DNA using energy provided by hydrolysis of ATP Direction of the sequence to the lagging strand is opposite the direction that helicase is unwinding the fork After remove the RNA primer, DNA polymerase 1 has extended the previous DNA and filled it in. Its going to have a nick next to the next DNA, every 1000 bases going to have a nick. DNA ligase (ATP or NAD is used as the cofactor) seals the nicks lots of nicks are points in recombination that promote lots of translocations and recom- bination events. So the cell doesn’t want lots of nicks in DNA, as soon as polymerase 1 causes the nick, ligase comes in and fills the nick. Path of replication Designated protein recognizes the origin in E. Coli, it is DNA A the protein in conjunction with another protein causes the DNA to melt. The initiating protein does this. Only the single strand region is the substrate for the helicase the helicase doesn’t start to melt, the initiating protein starts to melt the DNA then the helicase comes in and separates the strands In e. coli helicase is a hexameric repeat. in other organisms it could be other multimers Helicases bind both single and double stranded DNA in two different conforma- tions Enzymes have two different conformations stabilized when bound to ATP Stabilized when bound to ADP Every time you hydrolyze ATP one base pair is unwound by helicase
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The protein that melted the origin is not a replication fork because it doesn’t allow enough single strand to assemble any body else. You have to get the helicase to unwind with at least 16 base pairs to have a big enough single strand stretch to assemble the polymerase and all the other accessory proteins direct the helicase is unwinding, the one that creates the fork, is going in the direc-
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This note was uploaded on 01/26/2009 for the course BIOLOGY 244 taught by Professor Palter during the Fall '05 term at Temple.

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Lecture 10 - Lecture10...

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