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BIOL 239 Quiz: Set 11- DNA Replication

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Christine Dupont

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DNA Replication  The Watson-crick DNA replication model o This model proposed a mechanism whereby DNA replication is semiconservative: in daughter molecules one strand is conserved from the parental molecule and the other is newly synthesized o During replication the strands separate and used as templates to make two new daughter strands  What are the three possible models of DNA replication? o Semiconservative:  DNA molecules split and daughter molecules form one new strand one old strand o Conservative:  DNA molecule makes two new molecules by itself o Dispersive:  Parts of a new molecule is dispersively used to form old bits from new bits Error! Filename not specified.  Who proved DNA semi-conservation? How? o Matthew Meselson and Franklin Stahl Experimental proof of semiconservative replication o Studies with controlled isotopic composition of nucleotides incorporated into daughter DNA strands o Grew cells with N14 isotope and N15 isotope so they are incorporated into their DNA. o DNA was centrifuged so that heavier DNA (N15) was at the core of tube and lighter DNA (N14) was closer to the tip. o Hybrid DNA was in-between these two areas. o By looking at the banding patterns could rule out the conservative model as you would see two clear N15 and N14 bands o To rule out the semiconservative model of DNA replication they let the replicated strands to re-replicate and found that they saw ¼ of the progeny DNA hybrid and ¾ light (N14)  The molecular mechanism of DNA replication o Complex process which occurs at a precise moment is cell cycle o Two steps: initiation phase and elongation (copying) o E. coli DNA replication as a model  The reason E. coli is a very important model organism is because it is very similar  Difference between prokaryote and eukaryote DNA? o Prokaryotes: Small circular (binary fission) Eukaryotes: Large linear  Describe the Initiation phase o Eukaryotes have this at s phase o Origin of replication = OriC o OriC is at a site of A-T base pairs since hydrogen bonds on these bases are easier to pull apart from each other o Initiator proteins: Proteins that recognize the sequence and the origin and start the strand separation of the two strands o DNA Helicase: One one the first enzymes acting on the replication bubble and works to unwind the DNA strand and works in both directions of the replication bubble o When this bubble gets to a certain size as it gets bigger, DNA starts to base pair together as this is the energetically favorable reaction. o Single-stranded- DNA- binding proteins: Stabilize the single strands and stop them from re-pairing together  Elongation: o DNA polymerase (the enzyme that builds DNA) MUST have free 3’ OH to extend upon and requires a primer to begin synthesis o DNA primase: An enzyme that builds RNA primer which provides a free 3’ OH which can be extended on by DNA polymerase o As helicase continues to make the bubble bigger, DNA Polymerase III starts to add nucleotides to the 3’OH end of the new strands in the 5’ to 3’ direction o Leading strand: continuous synthesis happens here as the bubble opens up as DNA polymerase III synthesizes DNA in the 3’ to 5’ direction o Lagging strand: The discontinuous synthesis of DNA occurs on the lagging strand due to the direction of the synthesis of DNA polymerase III being in the 3’ to 5’ direction o Okazaki fragments: DNA- RNA strands that make up the lagging strands, which are small fragments of DNA due to the synthesis of the lagging strand o During elongation, the RNA primers are removed by DNA polymerase I o DNA polymerase 1 fills in the gap (still 5’ to 3’) o DNA ligase seals the 3’ OH and 5’ PO ni4ks by catalyzing the formation of phosphodiester bonds o This completes the formation of the lagging strand o This illustration shows the synthesis and replacement of RNA primers during replication of the lagging strand of DNA. o A short RNA strand is synthesized to provide a 3’-OH primer for DNA synthesis. The RNA primer is subsequently removed and replaced with DNA by the dual 5’→ 3’ exonuclease and 5’ → 3’ polymerase activities built into DNA polymerase I. DNA ligase then covalently closes the nascent DNA chain, catalyzing the formation of phosphodiester linkages between adjacent 3’-hydroxyls and 5’-phosphates. o The 5’ → 3’ exonuclease activity of DNA polymerase I excises the RNA primer, and, at the same time, the 5’ → 3’ polymerase activity of the enzyme replaces the RNA with a DNA chain by usin
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