The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends.
A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.
D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008854:Microscopy, Electron; D011938:Rec A Recombinases / Q000378:metabolism*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D015636:Magnesium Chloride; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D008274:Magnesium