Escherichia coli PriA Protein, Two Modes of DNA Binding and Activation of ATP Hydrolysis

Taku Tanaka,Toshimi Mizukoshi,Kaori Sasaki,Daisuke Kohda,Hisao Masai

doi:10.1074/jbc.m701848200

Abstract

Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3' terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3' terminus recognition. The former mode of binding requires the 3' terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.

Highlights

Escherichia coli PriA protein was originally identified as a component essential for conversion of the ␾X174 singlestranded genome to double-stranded replicative form
Other biochemical activities of PriA protein include ATP hydrolysis that strictly depends on binding to specific DNAs (7, 18) and 3Ј to 5Ј DNA helicase actions (19, 20)
PriA is a vital protein for cellular responses to replication fork blocks in bacterial cells, and it may serve as a sensor and stabilizer for the arrested replication forks (25–27)

Summary

Introduction

Escherichia coli PriA protein was originally identified as a component essential for conversion of the ␾X174 singlestranded genome to double-stranded replicative form. On A-fork [5Ј], feeble protection was detected on the unreplicated arm near the branch point (Ϫ1 to Ϫ6), which extended over the larger segment on both the lagging strand and unreplicated arms, when more PriA was added (Fig. 1E, lanes 1– 8), consistent with the lower affinity of PriA to this substrate in gel shift assays (12, 24).

Results

Conclusion