DNA replication initiation is mediated across all domains of life by initiator proteins oligomerizing at replication origins. Recently, it was shown that initiators can directly bind single-stranded DNA (ssDNA) and thus might enhance origin melting. In this study, we used single-molecule fluorescence assays to probe the ssDNA binding mechanism of the replication initiator DnaA. Our experiments revealed that DnaA forms a dynamic filament on ssDNA in 3' to 5' directionality in the presence of ATP and analogs. After nucleation with a three-monomer seed, monomers dynamically assemble and disassemble one monomer at a time at the 5' end, each monomer binding three nucleotides of ssDNA. The addition of adjacent double-stranded DnaA binding sites stabilized the DnaA filament on ssDNA. Our results extend the current models of origin melting via DnaA ssDNA interaction.
© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.