Effects of Conserved Wedge Domain Residues on DNA Binding Activity of RecG Helicase.

is extremely resistant to ionizing radiation and has an exceptional ability to repair DNA damage caused by various DNA-damaging agents. uses the same DNA-repair strategies as other prokaryotes, but certain proteins involved in the classical DNA repair machinery have characteristics different from their counterparts. RecG helicase, which unwinds a variety of branched DNA molecules, such as Holliday junctions (HJ) and D-loops, plays important roles in DNA repair, recombination, and replication. Primary sequence analysis of RecG from a number of bacterial species revealed that three amino acids (QPW) in the DNA-binding wedge domain (WD) are well-conserved across the RecG proteins. Interactions involving these conserved residues and DNA substrates were predicted in modeled domain structures of RecG (DrRecG). Compared to the WD of RecG protein (EcRecG) containing FSA amino acids corresponding to QPW in DrRecG, the HJ binding activity of DrRecG-WD was higher than that of EcRecG-WD. Reciprocal substitution of FSA and QPW increased and decreased the HJ binding activity of the mutant WDs, EcRecG-WD, and DrRecG-WD, respectively. Following -irradiation treatment, the reduced survival rate of DrRecG mutants (Δ) was fully restored by the expression of DrRecG, but not by that of EcRecG. EcRecG also enhanced -radioresistance of Δ, whereas DrRecG did not. Δ cells complemented by DrRecG and EcRecG reconstituted an intact genome within 3 h post-irradiation, as did the wild-type strain, but Δ with EcRecG and DrRecG exhibited a delay in assembly of chromosomal fragments induced by -irradiation. These results suggested that the QPW residues facilitate the association of DrRecG with DNA junctions, thereby enhancing the DNA repair efficiency of DrRecG.