RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins

J Mol Biol. 1997 Feb 7;265(5):519-40. doi: 10.1006/jmbi.1996.0748.

Abstract

RecA protein filaments formed on circular (ssDNA) in the presence of ssDNA binding protein (SSB) are generally stable as long as ATP is regenerated. On linear ssDNA, stable RecA filaments are believed to be formed by nucleation at random sites on the DNA followed by filament extension in the 5' to 3' direction. This view must now be enlarged as we demonstrate that RecA filaments formed on linear ssDNA are subject to a previously undetected end-dependent disassembly process. RecA protein slowly dissociates from one filament end and is replaced by SSB. The results are most consistent with disassembly from the filament end nearest the 5' end of the DNA. The bound SSB prevents re-formation of the RecA filaments, rendering the dissociation largely irreversible. The dissociation requires ATP hydrolysis. Disassembly is not observed when the pH is lowered to 6.3 or when dATP replaces ATP. Disassembly is not observed even with ATP when both the RecO and RecR proteins are present in the initial reaction mixture. When the RecO and RecR proteins are added after most of the RecA protein has already dissociated, RecA protein filaments re-form after a short lag. The newly formed filaments contain an amount of RecA protein and exhibit an ATP hydrolysis rate comparable to that observed when the RecO and RecR proteins are included in the initial reaction mixture. The RecO and RecR proteins thereby stabilize RecA filaments even at the 5' ends of ssDNA, a fact which should affect the recombination potential of 5' ends relative to 3' ends. The location and length of RecA filaments involved in recombinational DNA repair is dictated by both the assembly and disassembly processes, as well as by the presence or absence of a variety of other proteins that can modulate either process.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Bacterial Proteins / pharmacology
  • Cloning, Molecular
  • DNA Repair
  • DNA, Bacterial / metabolism
  • DNA, Bacterial / ultrastructure
  • DNA, Single-Stranded / metabolism*
  • DNA, Single-Stranded / ultrastructure
  • Deoxyadenine Nucleotides / pharmacology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Microscopy, Electron
  • Molecular Structure
  • Protein Binding
  • Rec A Recombinases / chemistry*
  • Rec A Recombinases / metabolism*
  • Rec A Recombinases / ultrastructure
  • Recombination, Genetic

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • DNA, Single-Stranded
  • Deoxyadenine Nucleotides
  • Escherichia coli Proteins
  • RecO protein, E coli
  • RecR protein, E coli
  • RecR protein, Bacteria
  • Rec A Recombinases
  • 2'-deoxyadenosine triphosphate