PS11KEY - PROBLEM SET ON CELL CYCLE IS ON THE WEB(2 PS...

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Professor Hinck Bio 110, Fall 2008, problem set 11 1) You are studying chromatin structure in rat liver DNA. When you digest rat liver nuclei briefly with micrococcal nuclease, extract the DNA, and run it on an agarose gel, it forms a ladder of broad bands spaced at about 200 nucleotide intervals. If you use the enzyme DNase I instead, there is a much more continuous smear of DNA on the gels with only the haziest suggestion of a 200 nucleotide repeat. If you denature the DNase I-treated DNA before fractionating it by gel electrophoresis, you find a new ladder of bands with a regular spacing of about 10 nucleotides. You are puzzled by the different results with these two enzymes. When you describe the experiments to the rest of your research group, one colleague suggests that the difference derives from the steric properties of the DNA binding sites on the two enzymes: micrococcal nuclease can only bind and cleave DNA that is free, whereas DNase I can bind and cut free DNA and DNA that is bound to the surface of a nucleosome. Your colleague predicts that if DNA is bound to any surface and digested with DNase I, it will generate a 10-nucleotide ladder. You test this prediction by binding DNA to polylysine-coated plastic dishes (just provides a positive charge for DNA to bind to) and digesting with the two enzymes. Micrococcal nuclease causes minimal digestion, but DNase I generates a 10- nucleotide ladder, verifying your friends suggestion. A) Why does brief digestion of nuclei with micrococcal nuclease yield a ladder of bands spaced at intervals of about 200 nucleotides? B) If you digested nuclei extensively with micrococcal nuclease, what pattern would you expect to see after fractionation of the DNA by gel electrophoresis? C) Explain how your colleague’s suggestion accounts for the generation of a 10- nucleotide ladder when nuclei are digested with DNaseI?
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