Stabilization of stalled replication forks by fork-binding proteins

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We previously showed that E. coli PriA protein, in collaboration with RecG, specifically binds and can stabilize an arrested replication fork. In this proposal we attempted to stabilize stalled replication forks in eukaryotic cells by utilizing these two proteins. We first developed an antibody against PriA and showed that stalled replication forks can be detected by ChIP-chip analyses. In response to fork arrest treatment, PriA bound to oriC in the wild-type cells, whereas it bound to the terC region under the condition where oriC is not functional. We then expressed PriA in fission yeast cells, examined its binding to chromatin. PriA was enriched near the replication origins. We are now expressing PriA in various mutant cells, and will examine if PriA can stabilize the fork by measuring the length of SSB coated single-strand.