Stabilization of a stalled replication fork by concerted actions of two helicases

Abstract

PriA helicase plays crucial roles in restoration of arrested replication forks. It carries a "3′ terminus binding pocket" in its N-terminal DNA binding domain, which is required for high affinity binding of PriA to a fork carrying a 3′-end of a nascent leading strand at the branch. We show that the abrogation of the 3′ terminus recognition either by a mutation in the 3′ terminus binding pocket or by the bulky modification of the 3′-end leads to unwinding of the unreplicated duplex arm on this fork, causing potential fork destabilization. This indicates a critical role of the 3′ terminus binding pocket of PriA in its "stable" binding at the fork for primosome assembly. In contrast, PriA unwinds the unreplicated duplex region on a fork without a 3′-end, potentially destabilizing the fork. However, this process is inhibited by RecG helicase, capable of regressing the fork until the 3′-end of the nascent leading strand reaches the branch. PriA now stably binds to this regressed fork, stabilizing it. Using a model arrest-fork-substrate, we reconstitute the above process in vitro with RecG and PriA proteins. Our results present a novel mechanism by which two helicases function in a highly coordinated manner to generate a structure in which an arrested fork is stabilized for further repair and/or replication restart. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.