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BIOB11H3 (13)
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Chapter 13

Chapter 13 notes

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

Chapter 13 Watson and Crick hypothesized that DNA replication is semiconservative - but they also had to consider the conservative and dispersive modes of replication as alternative hypotheses. Meselson-Stahl tested this hypothesis by growing DNA in a medium with heavy nitrogen isotope (15N) - then removing it and placing it in a light nitrogen medium (14N) - then took samples and subjected them to equilibrium density-gradient centrifugation in cesium chloride solution at different times: - If conservative, there should always be a heavy band - light band starts with the same amount, then increases everytime. - If dispersive, first only hybrid band - then the whole band starts moving up towards the light region while increasing. - If semiconservative, hybrid band should always be there and light band starts to increase from 0. Taylor showed that semiconservative replication also occur in eukaryotes. Cells were put in medium with bromodeoxyuridine (BrdU) that is incorporated into DNA in place of thymidine and could be contrasted with others after staining. After 2 generations, he found that one chromatid of each chromosome was composed of 2 BrdU-containing strands, whereas the other chromatid was a hybrid. Separation of DNA molecules creates topological problems (torsional stress). An overwound DNA becomes positively supercoiled ahead of the www.notesolution.com fork. DNA gyrase (type II topoisomerase) relieves the mechanical strain during replication - they travel along the DNA ahead of the replication fork and removes positive supercoiling by cleaving and sealing. DNA must have specific structural requirements to initiate replication. DNA polymerase cannot initiate the formation of DNA strand - it can only add nucleotides to a 3’ hydroxyl terminus of an existing strand. The strand that provides this is called a primer. DNA polymerase III is the major enzyme responsible for replication, but there’s only 10 copies for every 300-400 polymerase I. New DNA strand are always synthesized 5’-3’ direction. So, there’s leading and lagging strand. Replication is semidiscontinuous. Okazaki found that if he incubated bacteria in 3H thymine, after a few seconds most of the labeled DNA was short, but after a few minutes most of it was large. This suggested that a portion of the DNA was constructed in small segments and then joined by DNA ligase. Initiation is accomplished by a type of RNA polymerase called primase, which constructs a short RNA primer required for initiation. At the replication fork, DNA helicase uses ATP to unwind DNA by moving along one of the DNA strands and breaking hydrogen bonds. The major one is DnaB, which first binds to origin of replication by the help of DnaC, and then proceeds. DNA unwinding is aided by the www.notesolution.com attachment of single stranded DNA-binding (SSB) proteins to the separated DNA strands to prevent them from rewinding. In bacteria, primase and helicase associate together to form primosome. The same DNA polymerase III synthesizes all lagging strands. It moves along with the DNA polymerase III that is synthesizing the leading strand - they are both a part of a single protein complex. This is accomplished by causing the lagging strand to form a loop, so that both would still be synthesizing in the 5’-3’ direction even though thy are moving together along the DNA. DNA polymerase III is a part of DNA polymerase III holoenzyme (or replisome). Another component is b clamp - keeps polymerase associated with the DNA template. The assembly of b clamp with the DNA molecules requires a clamp loader, which is another part of the replisome. Once the DNA enters the b clamp, the ATP on the loader is hydrolyzed and and openning of the clamp closes. When DNA polymerase synthesizes an Okazaki fragment, it disengages from the b clamp and binds to another one. DNA polymerase I is responsible for repairing and replacing RNA primers on Okazaki fragments with DNA. It has 3’-5’ and 5’-3’ exonucleases, in addition to its polymerizing activity. The 5’-3’ exonuclease removes RNA primers and its polymerizing activity adds DNA in its place. www.notesolution.com If the incoming nucleotide does not form the proper geometry with the other nucleotide then the fingers of DNA polymerase I cannot fold onto the palm, and so, cannot catalyse the reaction to link the two (conformational change is required for the enzymatic activity). When incor
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