The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A.
Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.
Eggler
Aimee L
AL
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Inman
Ross B
RB
Cox
Michael M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D008957:Models, Genetic; D009711:Nucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051135:Rad51 Recombinase; D051716:Replication Protein A; D013997:Time Factors
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009711:Nucleotides; D009841:Oligonucleotides; D051716:Replication Protein A; D000255:Adenosine Triphosphate; D004247:DNA; D051135:Rad51 Recombinase