Abstract
E. coli RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments, as the first step of this repair process. It has been suggested that RecA filaments assemble mainly by binding and extending onto the free ssDNA region not covered by SSB, or are assisted by mediators. Using the tethered particle motion (TPM) technique, we monitored individual RecA filament assembly on SSB-wrapped ssDNA in real-time. Nucleation times of the RecA E38K nucleoprotein filament assembly showed no apparent dependence among DNA substrates with various ssDNA gap lengths (from 60 to 100 nucleotides) wrapped by one SSB in the (SSB)65 binding mode. Our data have shown an unexpected RecA filament assembly mechanism in which a RecA-SSB-ssDNA interaction exists. Four additional pieces of evidence support our claim: the nucleation times of the RecA assembly varied (1) when DNA substrates contained different numbers of bound SSB tetramers; (2) when the SSB wrapping mode conversion is induced; (3) when SSB C-terminus truncation mutants are used; and (4) when an excess of C-terminal peptide of SSB is present. Thus, a RecA-SSB interaction should be included in discussing RecA regulatory mechanism.
Highlights
Dynamics of RecA filament assembly in the absence of other DNA binding proteins has been investigated by both conventional, ensemble-based biochemical experiments as well as various single-molecule ones[1,9,10,11,12,13,14,15]
In the absence of single-stranded DNA binding proteins (SSB), wild-type RecA (wtRecA) assembled into filaments on about 4.5% of the DNA substrates within 2.5 minutes of observation, while 35% of the DNA tethers formed filaments with the E38K mutants (Figure S1A–F)
In addition to nucleation on free singlestranded DNA (ssDNA), we showed that there exists an additional pathway that RecA filaments assemble through a RecA-SSB interaction
Summary
Dynamics of RecA filament assembly in the absence of other DNA binding proteins has been investigated by both conventional, ensemble-based biochemical experiments as well as various single-molecule ones[1,9,10,11,12,13,14,15]. Previous studies have suggested a passive nucleation model during RecA filament formation on SSB-wrapped ssDNA2, where RecA nucleation takes place in the presence of a transiently exposed, free ssDNA segment not covered by SSB. Experimental modifications using either a pre-assembled RecA-ATPγS-dsDNA nucleus to significantly enhance the assembly process[12,24] or taking only snapshots of dye-labeled RecA on SSB-bound kilo-nucleotide ssDNA2 were used These approaches compromised either temporal (pre-assembled RecA-dsDNA filament) or spatial information (diffraction-limited spot of RecA nucleation cluster landing onto a ssDNA without defined numbers of bound SSB molecules) of how RecA initiates filament formation on SSB-wrapped ssDNA. A single-molecule tool with a time window of tens of minutes in real-time and a precise control of bound SSB numbers are required
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