RecFOR proteins target RecA protein to a DNA gap with either DNA or RNA at the 5' terminus: implication for repair of stalled replication forks.
The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1-2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000-2000 nucleotides downstream (5' → 3') of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5'-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.
Department of Microbiology and of Molecular and Cellular Biology, University of California, Davis, California 95616.
Katsumi
K
Morimatsu
Department of Microbiology and of Molecular and Cellular Biology, University of California, Davis, California 95616.
Yun
Y
Wu
Department of Microbiology and of Molecular and Cellular Biology, University of California, Davis, California 95616. Electronic address: sckowalczykowski@ucdavis.edu.
Stephen C
SC
Kowalczykowski
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C081006:RecO protein, E coli; C489340:recF protein, E coli; C065831:RecR protein, Bacteria; D011938:Rec A Recombinases