title: The PriA replication restart protein blocks replicase access prior to helicase assembly and directs template specificity through its ATPase activity.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80303, USA.
authors.forename: Carol M
authors.initials: CM
authors.lastname: Manhart
authors.forename: Charles S
authors.initials: CS
authors.lastname: McHenry
mesh_terms: D019943:Amino Acid Substitution; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D020125:Mutation, Missense; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C476430:SSB protein, E coli; C506743:priA protein, E coli; D004265:DNA Helicases