The efficiency of strand invasion by Escherichia coli RecA is dependent upon the length and polarity of ssDNA tails.
RecA protein is essential for homologous recombination and the repair of DNA double-strand breaks in Escherichia coli. The protein binds DNA to form nucleoprotein filaments that promote joint molecule formation and strand exchange in vitro. RecA polymerises on ssDNA in the 5'-3' direction and catalyses strand exchange and branch migration with a 5'-3' polarity. It has been reported previously, using D-loop assays, in which ssDNA (containing a heterologous block at one end) invades supercoiled duplex DNA that 3'-homologous ends are reactive, whereas 5'-ends are inactive. This polarity bias was thought to be due to the polarity of RecA filament formation, which results in the 3'-ends being coated in RecA, whereas 5'-ends remain naked. Using a range of duplex substrates containing ssDNA tails of various lengths and polarities, we now demonstrate that when no heterologous block is imposed, 5'-ends are just as reactive as 3'-ends. Moreover, using short-tailed substrates, we find that 5'-ends form more stable D-loops than 3'-ends. This bias may be a consequence of the instability of short 3'-joints. With more physiological substrates containing long ssDNA tails, we find that RecA shows no intrinsic preference for 5' or 3'-ends and that both form D-loop complexes with high efficiency.
McIlwraith
M J
MJ
Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, Hertfordshire, EN6 3LD, UK.
West
S C
SC
D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases