This proposal focuses on the mechanisms of replication of simple DNA repeats in vivo. Changes in the length of such repeats are responsible for numerous human neurological diseases and some forms of cancer. It is believed that these changes are caused by abnormal replication of the repetitive tracts, but direct data supporting this notion are scarce. Using electrophoretic analysis of replication intermediates as a tool and bacterial plasmids as a model system, the principal investigator has directly shown that progression of the replication fork through several repetitive DNA stretches is unusually slow and the lagging DNA strand of the repeat is often underreplicated. He has found that three different mechanisms account for the replication stalling caused by different repeats. One mechanism is likely to be a stable secondary structure of the repeated DNA situated on the lagging strand template. Another is due to transcription stalling at the repeated DNA stretch followed by replication blockage. Finally, cooperative protein binding to the third group of repeats is responsible for replication attenuation. The principal investigator hypothesizes that replication stalling could be responsible for the repeat-length instability. In this proposal, he will focus on the detailed characterization of these mechanisms in yeast and bacterial systems. Specifically, he will study the replication of trinucleotide repeats in a yeast experimental system using electrophoretic analysis of replication intermediates. Using gene replacement methods, he will obtain different mutants of the yeast replication apparatus in order to determine the components affecting the replication fork progression through DNA repeats. The principal investigator will then study the correlation between changes in the replication of trinucleotide repeats and their expansion rates in yeast replicative mutants. He will also study the relation between transcription and replication-stalling within homopurine-homopyrimidine repeats in the E. coli and yeast systems. He will determine the fine structure of stalled intermediates in vivo and in vitro using mutational analysis and chemical footprinting. Finally, he will study the mechanism of replication fork attenuation caused by protein binding to repetitive DNA stretches. To this end, he will purify and characterize the novel E. coli protein that binds specifically to d(G-A)n d(T-C)n repeats. The long-term goal is to understand the role played by replication of simple DNA repeats in their maintenance and length polymorphism.
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