Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity.
recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D002678:Chimera; D003090:Coliphages / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D007700:Kinetics; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D000244:Adenosine Diphosphate; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I