pset1_ans

pset1_ans - MIT Department of Biology 7.28, Spring 2005 -...

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MIT Department of Biology 7.28, Spring 2005 - Molecular Biology Question 1 You are studying the mechanism of DNA polymerase loading at eukaryotic DNA replication origins. Unlike the situation in bacteria, the three different eukaryotic DNA polymerases are not tightly associated with one another and you are interested in how they are all brought to the origin to assemble the replication fork. Your ability to address this question is greatly facilitated by the recent development of a completely purified set of proteins that will initiate DNA replication from an S. cerevisiae (Baker’s yeast) origin of replication. Your first goal is to determine the order of assembly of the 3 DNA polymerases during initiation. (A) Describe an assay you would use to monitor the assembly of the DNA polymerases at the origin. You have access to all the purified proteins required for replication initiation including the three purified DNA polymerases in labeled and unlabeled forms. A gel filtration assay can be used to measure the assembly of DNA polymerases at the origin. This assay would require an origin containing plasmid DNA and 32P- labeled DNA polymerases, which are incubated with the DNA to allow loading. To measure assembled polymerases, one must separate bound (to DNA) polymerases from unbound (free) by using gel filtration (bound polymerases will elute first with DNA, whereas free will elute later in the fractions). In order to determine whether protein is DNA associated or free, the amount of radioactivity present in the fractions is determined by using a scintillation counter, which will indicate the presence of the labeled polymerase. By performing this analysis with all combinations of polymerases alone and with other polymerases, each time labeling the polymerase one is assaying with 32P, one can determine which polymerase(s) can load by themselves, which can load in the presence of other polymerases, as well as the relative order of loading. Using your assay, you determine what effect leaving out one polymerase has on the assembly of the other two DNA polymerases at the origin. You obtain the following results: DNA Polymerases Added Pol α /Primase, Pol ε Pol α /Primase, Pol δ Pol δ , Pol ε Pol α /Primase, Pol δ , Pol ε DNA Polymerases Assembled Pol ε Pol δ Pol δ , Pol ε Pol α /Primase, Pol δ , Pol ε
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(B) What can you conclude about the order of loading of the three DNA polymerases based on these data? Briefly explain the logic behind your conclusions. In order for the Pol α /Primase to load, both Pol ε and Pol δ must be already loaded. Pol ε and Pol δ can load independently of one another and Pol α /Primase. Pol α /Primase only loads in the last experiment when both Pol ε and Pol δ are present, and cannot load in the first two lanes when only one or the other is present. The simplest hypothesis is that Pol
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pset1_ans - MIT Department of Biology 7.28, Spring 2005 -...

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