RecA protein dynamics in the interior of RecA nucleoprotein filaments.
We characterize aspects of the conformation and dynamic state of RecA filaments when bound to dsDNA that are specifically linked to the presence of the second of the two bound DNA strands. Filaments bound to dsDNA exhibit a facile exchange between free and bound RecA monomers or oligomers in the filament interior that is not seen on ssDNA. The RecA mutant K72R, which binds but does not hydrolyze ATP, forms mixed filaments with wild type RecA protein under some conditions. In the presence of dATP, mixed filaments are formed on dsDNA or ssDNA in which the RecA K72R content approximately reflects the proportion of the K72R mutant in the total RecA protein present when the filament is formed. In the presence of ATP, mixed filaments are formed on dsDNA, but the mutant protein strongly inhibits the binding of wtRecA protein to single-stranded DNA. When RecA K72R is added to pre-formed filaments containing only wild-type RecA protein on single-stranded DNA, little of the mutant protein exchanges into the filament. Exchange occurs readily, however, when the filament is bound to double-stranded DNA. The presence of a second DNA strand in RecA-dsDNA filaments produces as altered and more dynamic filament state relative to filaments formed on single-stranded DNA. The results point to a substantial alteration in filament state when synapsis occurs during RecA protein-mediated DNA strand exchange.
Shan
Q
Q
Department of Biochemistry, University of Wisconsin-Madison, 53706, USA.
Cox
M M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008957:Models, Genetic; D008968:Molecular Conformation; D009154:Mutation; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D009711:Nucleotides; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases