A RecA Mutant, RecA730, Suppresses the Recombination Deficiency of the RecBC1004D-χ* Interaction in Vitro and in Vivo

Naofumi Handa, Stephen C. Kowalczykowski

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

In Escherichia coli, homologous recombination initiated at double-stranded DNA breaks requires the RecBCD enzyme, a multifunctional heterotrimeric complex that possesses processive helicase and exonuclease activities. Upon encountering the DNA regulatory sequence, χ, the enzymatic properties of RecBCD enzyme are altered. Its helicase activity is reduced, the 3′→5′nuclease activity is attenuated, the 5′→3′ nuclease activity is up-regulated, and it manifests an ability to load RecA protein onto single-stranded DNA. The net result of these changes is the production of a highly recombinogenic structure known as the presynaptic filament. Previously, we found that the recC1004 mutation alters χ-recognition so that this mutant enzyme recognizes an altered χ sequence, χ*, which comprises seven of the original nucleotides in χ, plus four novel nucleotides. Although some consequences of this mutant enzyme-mutant χ interaction could be detected in vivo and in vitro, stimulation of recombination in vivo could not. To resolve this seemingly contradictory observation, we examined the behavior of a RecA mutant, RecA730, that displays enhanced biochemical activity in vitro and possesses suppressor function in vivo. We show that the recombination deficiency of the RecBC1004D-χ* interaction can be overcome by the enhanced ability of RecA730 to assemble on single-stranded DNA in vitro and in vivo. These data are consistent with findings showing that the loading of RecA protein by RecBCD is necessary in vivo, and they show that RecA proteins with enhanced single-stranded DNA-binding capacity can partially bypass the need for RecBCD-mediated loading.

Original languageEnglish (US)
Pages (from-to)1314-1325
Number of pages12
JournalJournal of Molecular Biology
Volume365
Issue number5
DOIs
StatePublished - Feb 2 2007

Keywords

  • DNA repair
  • RecA
  • RecA-loading
  • RecBCD
  • recombination hotspot

ASJC Scopus subject areas

  • Virology

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