DNA wrapped in the nucleosome is sterically occluded, yet must be accessed for vital cellular processes, including transcription, replication, recombination, and DNA repair. We are studying spontaneous and also ATP dependent processes that provide access to buried stretches of nucleosomal DNA.
Studies of spontaneous accessibility have focused on characterizing the nucleosome
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conformational changes that make
buried sites accessible, and the frequencies of these conformational
changes. We find that the mechanism of spontaneous accessibility is
via partial DNA unwrapping starting from one end of the nucleosome
and proceeding inward. Spontaneous unwrapping occurs rapidly (>>1
sec-1) and with no loss or exchange of histones. Our studies of the ATP-dependent processes, conducted in collaboration with the Becker group (ABI, Munich), focus on the Drosophila nucleosome remodeling factor ISWI and it's complex with Acf-1 (ACF). Studies of the enzyme’s ATPase activity reveal that ISWI, and ACF are not active dimers as predicted by their cooperative binding to DNAs, but rather are active monomers, with a preferred substrate of chromatin over individual nucleosomes or naked DNA. Future work will focus on quantitative measurements to shed light on the mechanistic steps of ISWI and ACF induced nucleosome movement. |
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