Uncoupling of the ATPase activity from the branch migration activity of RuvAB protein complexes containing both wild-type and ATPase-defective RuvB proteins.
BACKGROUND
Escherichia coli RuvAB promotes branch migration of Holliday junctions during recombination repair and homologous recombination. RuvB forms a hexameric ring through which duplex DNA passes and is translocated in an ATP-dependent manner. ATPase-deficient RuvB mutant K68A has a mutation in the Walker A motif and exerts a dominant-negative effect on in vivo repair of UV-induced DNA damage. In this study, we examined RuvAB-dependent branch migration in the presence of a mutant RuvB, K68A.


RESULTS
Mixing K68A with wild-type RuvB resulted in the formation of heterohexamers that showed unique properties of DNA binding, ATPase, and branch migration activities different from those of either wild-type or mutant homohexamers. RuvB heterohexamers inhibited branch migration and caused Holliday junctions to accumulate during RecA-mediated strand exchange. In the presence of RuvA, RuvB heterohexamers had Holliday junction-dependent ATPase activity, but did not promote branch migration.


CONCLUSIONS
These results suggest that functional cooperation among the subunits in the hexamers is required for branch migration, but inclusion of inactive subunits is tolerated for ATP hydrolysis. Therefore, we propose that an essential ATP hydrolysis-dependent functional cooperation is induced in RuvB hexamer subunits during RuvAB-mediated branch migration.
Hishida
Takashi
T
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Iwasaki
Hiroshi
H
Han
Yong-Woon
YW
Ohnishi
Takayuki
T
Shinagawa
Hideo
H
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009154:Mutation; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C061260:RuvB protein, Bacteria; D011938:Rec A Recombinases; C071059:Holliday junction DNA helicase, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases