Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Peter McGlynn, R. G. Lloyd, K. J. Marians

Research output: Contribution to journalArticle

130 Citations (Scopus)

Abstract

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecC, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecC promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecC, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

Original languageEnglish
Pages (from-to)8235-8240
Number of pages5
JournalPNAS
Volume98
Issue number15
DOIs
Publication statusPublished - Jul 2001

Keywords

  • ESCHERICHIA-COLI
  • TOPOISOMERASE-IV
  • PROTEIN
  • STRAND
  • PURIFICATION
  • RESOLVASE
  • IDENTIFICATION
  • POLYMERASE
  • UNLINKING
  • MECHANISM

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