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 pres ence of the second of the two bound DNA strands. Filaments bound to ds DNA 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 fila ments with wild type RecA protein under some conditions. In the presen ce of dATP, mixed filaments are formed on dsDNA or ssDNA in which the RecA K72R content approximately reflects the proportion of the K72R mu tant in the total RecA protein present when the filament is formed. In the presence of ATP, mixed filaments are formed on dsDNA, but the mut ant protein strongly inhibits the binding of wtRecA protein to single- stranded DNA. When RecA K72R is added to pre-formed filaments containi ng only wild-type RecA protein on single-stranded DNA, little of the m utant protein exchanges into the filament. Exchange occurs readily, ho wever, when the filament is bound to double-stranded DNA. The presence of a second DNA strand in RecA-dsDNA filaments produces as altered an d more dynamic filament state relative to filaments formed on single-s tranded DNA. The results point to a substantial alteration in filament state when synapsis occurs during RecA protein-mediated DNA strand ex change. (C) 1996 Academic Press Limited