Identification of Subunit Binding Positions on a Model Fork and Displacements that Occur During Sequential Assembly of the E. coli Primosome [Enzymology]

March 5th, 2015 by Manhart, C. M., McHenry, C. S.

When replication stalls and forks disassemble, the restart primosome is required to reload the replicative helicase so that chromosomal replication can be reinitiated. We have taken a photo-crosslinking approach, using model replication forks containing a phenyl diazirine placed at single locations, to determine the positions of primosomal protein binding and changes in interactions that occur during the assembly reaction. This approach revealed a novel mode for SSB*-DNA binding where SSB interacts with both the leading and lagging single-strand segments and the parental duplex of the fork. Crosslinking to a novel region within SSB is observed only when it is bound to forked structures. This binding mode is also followed by PriB. PriA binds to the fork, excluding SSB and PriB, interacting with the primer terminus, single-stranded leading and lagging strands and duplex in immediate proximity of the fork. SSB binds to flanking single-stranded segments distal to the fork in the presence of PriA. Addition of PriB or DnaT to a PriA-SSB-fork complex does not lead to cross-linking or displacement suggesting their association is through protein-protein interactions at early stages of the reaction. Upon addition of DnaC and the DnaB helicase in the presence of ATPĪ³S, helicase is assembled, leading to contacts within the duplex region on the tracking (lagging) strand and strong contacts with the displaced leading single strand near the fork. PriA is displaced from DNA upon helicase assembly.
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