The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity

Carol M Manhart,Charles S Mchenry

doi:10.1074/jbc.m112.435966

Abstract

The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. We have developed a primosomal protein-dependent fluorescence resonance energy transfer (FRET) assay using a minimal fork substrate composed of synthetic oligonucleotides. We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5'-ends, an event that can be blocked by attaching a steric block to the 5'-end or coating DNA with single-stranded DNA binding protein. The specificity of PriA for replication forks is regulated by its intrinsic ATPase. ATPase-defective PriA K230R shows a strong preference for substrates that contain no gap between the leading strand and the duplex portion of the fork, as demonstrated previously. Wild-type PriA prefers substrates with larger gaps, showing maximal activity on substrates on which PriA K230R is inactive. We demonstrate that PriA blocks replicase function on forks by blocking its binding.

Highlights

PriA initiates restart at stalled replication forks
We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5؅-ends, an event that can be blocked by attaching a steric block to the 5؅-end or coating DNA with single-stranded DNA binding protein
Development of a fluorescence resonance energy transfer (FRET) Assay for Primosome Function—We adapted a FRET-based assay for helicase function [22] and optimized it for studying the helicase loading apparatus from two representative Gram-negative and Gram-positive organisms, E. coli and B. subtilis

Summary

Background

PriA initiates restart at stalled replication forks. Results: A FRET assay detects E. coli and B. subtilis PriA-mediated helicase loading. The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. PriA appears to be the lead protein that directs assembly of the restart primosome This is consistent with its substrate specificity in binding to D loops, but not bubbles, and three-stranded structures that provide models for replication forks [7, 8]. It has been suggested that the primosome assembly process is a dynamic one in which these proteins hand off the fork substrate to one another, culminating with DnaT binding to PriB and displacing it from DNA to provide a landing site for the helicase loader and helicase [14]. Many of the basic principles of fork dynamics have been

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

The starting conditions used were as follows

DISCUSSION