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.
Highlights
In bacteria, DEXH/DEAD-type helicases have been implicated in cellular responses to replication fork arrest (18 –20)
On the basis of the in vitro studies using a novel arrest-fork-substrate, we propose a novel mechanism of stabilization and restart of arrested replication forks by the concerted actions of two helicases
A number of studies have been conducted on binding and helicase actions of PriA on various DNAs. It binds to structures mimicking D-loop or arrested replication forks in vitro (24, 25, 29 –32)
Summary
Proteins—Wild-type and mutant PriA proteins and RecG protein were expressed and purified as previously described (34 –37). Substrates termed [3Ј-P] carry the leading strand whose 3Ј-end is phosphorylated. The 5Ј-end of the oligonucleotide indicated was phosphorylated by T4 polynucleotide kinase (New England Biolabs) and [␥-32P]ATP prior to annealing. Each substrate was purified from polyacrylamide gel as described previously [34]. To prepare the partially complementary arrested fork (pcA-fork2 [3Ј, 5Ј] 49 L10), leading and lagging arms were independently annealed as described above. After isolation from polyacrylamide gel, both partial duplex constructs were mixed at room temperature for 4 h, and annealed arms were reisolated from polyacrylamide gel
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