Functional Integration of the Bacteriophage T4 DNA Replication Complex: The Multiple Roles of the ssDNA Binding Protein (gp32) #MMPMID41359173
Albrecht CS; Israels B; Maurer J; H von Hippel P; Marcus AH
Biochemistry 2025[Dec]; ? (?): ? PMID41359173show ga
Single-stranded (ss) DNA binding protein (gp32) serves as the central regulatory component of the multisubunit T4 bacteriophage DNA replication system by coordinating the system's three functional subassemblies, resulting in phage DNA synthesis in T4-infected Escherichia coli cells at the high speeds ( approximately 1000 nts s(-1)) and the high fidelity (<1 error per 10(7) nts) required for genomic function within this cellular ecosystem. Gp32 proteins continuously bind to, slide on as cooperatively linked clusters, and unbind from transiently exposed single strands of DNA to carry out their coordinating functions. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within gp32 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 Forster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine single strands of varying strand length and polarity near model ss-dsDNA [3',5'-oligo-(dT)(14,15)-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 strand polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTDs might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32 nucleoprotein filaments involved in phage DNA replication and related processes.
Biochemistry 2025 ; ? (?): ?
