The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in Bacillus subtilis.
In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in DeltaruvAB and DeltarecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct DeltaruvAB DeltarecG, DeltarecU DeltaruvAB, DeltarecU DeltarecG, or DeltarecU DeltarecJ strains. However, DeltaruvAB could be combined with addAB (recBCD), recF, recH, DeltarecS, DeltarecQ, and DeltarecJ mutations. The DeltaruvAB and DeltarecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a DeltarecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.
Sanchez
Humberto
H
Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Stockton-on-Tees TS17 6BH, United Kingdom.
Kidane
Dawit
D
Reed
Patricia
P
Curtis
Fiona A
FA
Cozar
M Castillo
MC
Graumann
Peter L
PL
Sharples
Gary J
GJ
Alonso
Juan C
JC
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D004249:DNA Damage / Q000502:physiology*; D004265:DNA Helicases / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism* / Q000528:radiation effects; D045566:DNA, Cruciform / Q000502:physiology / Q000528:radiation effects; D004706:Endodeoxyribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D045565:Holliday Junction Resolvases / Q000502:physiology*; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D045566:DNA, Cruciform; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C061260:RuvB protein, Bacteria; C069806:ruvC protein, E coli; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; D045565:Holliday Junction Resolvases; C047879:RecN protein, Bacteria; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases