title: 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.
authors.affiliation: Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Tanaka
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000502:physiology*; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D008956:Models, Chemical; D008957:Models, Genetic; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C071446:RecG protein, E coli; D000255:Adenosine Triphosphate; D004247:DNA; D003851:Deoxyribonucleases; D003850:Deoxyribonuclease I; D000251:Adenosine Triphosphatases; C506743:priA protein, E coli; D004265:DNA Helicases