EXCEED THE SPACE PROVIDED. Arrest of replication fork progression evokes a concerted cellular response in both prokaryotes and eukaryotes that is designed to repair whatever template damage that exists and restart replication. In bacteria, failure to restart replication is a lethal event. In eukaryotes, mutation of many of the proteins involved in recombinational repair of double-strand breaks that occur at stalled replication forks leads to cancer-predisposition syndromes. Thus, understanding the mechanisms of replication restart, which is the goal of our studies, is of considerable significance. In the previous grant period we made substantive progress in understanding the basis for nascent strand regression, the mechanism of double-strand break repair by recombination-directed replication, and the biochemical pathways of restart. In the extended grant period, we will continue our efforts to understand the mechanisms of restart, the interplay between nascent strand regression and restart, and the fate of the components of the replisome at stalled forks. In addition, we will develop new assays that allow us to study replisome remodeling and the competition between various recombination and replication pathways onjoint molecules. We will also initiate single molecule analyses of the action of the replication fork and the replicative helicase that will allow us to probe the nature of fork progression, the characteristics and causes of replication fork pausing, and the response of the replisome to roadblocks in the template. We also expect that the use of our new linear template will allow us to detect Origin-independent replisome-loading activities in eukaryotic extracts.
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