BRANCH MIGRATION OF HOLLIDAY JUNCTIONS - IDENTIFICATION OF RECG PROTEIN AS A JUNCTION SPECIFIC - DNA HELICASE

The product of the recG gene of Eschevichia coli is needed for normal recombination and DNA repair in E.coli and has been shown to help proc ess Holliday junction intermediates to mature products by catalysing b ranch migration. The 76 kDa RecG protein contains sequence motifs cons erved in the DExH family of helicases, suggesting that it promotes bra nch migration by unwinding DNA. We show that RecG does not unwind blun t ended duplex DNA or forked duplexes with short unpaired single-stran d ends. It also fails to unwind a partial duplex (52 bp) classical hel icase substrate containing a short oligonucleotide annealed to circula r single-stranded DNA. However, unwinding activity is detected when th e duplex region is reduced to 26 bp or less, although this requires hi gh levels of protein, The unwinding proceeds with a clear 3' to 5' pol arity with respect to the single strand bound by RecG. Substantially h igher levels of unwinding are observed with substrates containing a th ree-way duplex branch. This is attributed to RecG's particular affinit y for junction DNA which we demonstrate would be heightened by single- stranded DNA binding protein iii vivo. Reaction requirements for unwin ding 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 migra tion of Holliday junctions.