Specific mutations within the AT-rich region of a plasmid replication origin affect either origin opening or helicase loading

Prokaryotic and eukaryotic replicons possess a distinctive region containing a higher than average number of adenine and thymine residues (the AT-rich region) where, during the process of replication initiation, the initial destabilization (opening) of the double helix takes place. In many prokaryotic origins, this region consists of repeated 13-mer motifs whose function has not yet been specified. Here we identify specific mutations within the 13-mer sequences of the broad-host-range plasmid RK2 that can result in defective origin opening or that do not affect opening but induce defects in helicase loading. We also show that after the initial recruitment of helicase at the DnaA-box sequences of the plasmid origin, the helicase is translocated to the AT-rich region in a reaction requiring specific sequence of the 13-mers and appropriate facing of the origin motifs. Our results demonstrate that specific sequences within the AT-rich region of a replication origin are required for either origin opening or helicase loading