Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.
The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.
Whitby
M C
MC
Department of Genetics, University of Nottingham, Queens Medical Centre, UK.
Vincent
S D
SD
Lloyd
R G
RG
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic / Q000235:genetics*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C071446:RecG protein, E coli; D004265:DNA Helicases