Characterization of the ATPase Activity of the Escherichia coli RecG Protein Reveals that the Preferred Cofactor is Negatively Supercoiled DNA

Stephen L. Slocum, Jackson A. Buss, Yuji Kimura, Piero R. Bianco

Research output: Contribution to journalArticlepeer-review

45 Scopus citations

Abstract

RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.

Original languageEnglish (US)
Pages (from-to)647-664
Number of pages18
JournalJournal of Molecular Biology
Volume367
Issue number3
DOIs
StatePublished - Mar 30 2007
Externally publishedYes

Keywords

  • DNA helicase
  • DNA repair
  • RecG
  • recombination
  • replication fork reversal

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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