Dna Replication Initiation Studied at the Single Molecule Level

DNA replication initiation is mediated by initiator proteins across all domains of life. Initiator proteins form oligomers at replication origin sites and melt the AT-rich region to enable the replisome assembly. The origin melting by the initiator proteins is supposed to be enhanced by the interaction with single-stranded DNA. In this study, we use single-molecule FRET together with hidden Markov modeling to probe the single-stranded DNA binding mechanism of the Aquifex aeolicus replication initiator DnaA. We find that in the presence of the ATP-analog ADP•BeF3, the DnaA oligomer assembly and disassembly occurs one monomer at a time. Our FRET measurements further indicate a curly filament of DnaA monomers on ssDNA that supports the model of negative super-helical strain promoting efficient melting of the AT-rich region. We further test ssDNA binding with an ATP-independent replication initiator G38P from bacteriophage SPP1 and find as well a dynamic assembly and disassembly on ssDNA.