Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32)
Abstract
Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the system's three functional sub-assemblies, resulting in phage DNA synthesis in T4-infected E. coli cells at the high speeds (~1,000 nts s-1) and the high fidelity (< 1 error per 107 nts) required for genomic function within this cellular eco-system. Gp32 proteins continuously bind to, slide as cooperatively-linked clusters on, and un-bind from transiently exposed single-stranded (ss) DNA templates to carry out their coordinating functions, as well as to protect genomic sequences from nuclease activity and block the formation of interfering secondary structures. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within ssb clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule Förster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine lattices of varying lattice length and polarity near model ss-dsDNA junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by…
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