Israels, B. | Search bioRxiv

bioRxiv2025

Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32)

Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the systems 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 Forster 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 lattice polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at lattice positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTD domains might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32-ssDNA filaments involved in phage DNA replication and related processes.

biochemistry↗

bioRxiv2021

Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

Thermally-driven conformational fluctuations (or breathing) of DNA plays important roles in the function and regulation of the macromolecular machinery of genome expression. Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here we use microsecond-resolved single-molecule Forster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short oligothymidine [oligo(dT)n] templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond time-scales, between three macrostates with distinctly different end-to-end distances. These are: (i) a compact macrostate that represents the dominant species at equilibrium; (ii) a partially extended macrostate that exists as a minority species; and (iii) a highly extended macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA associating proteins. Significance StatementThe genetic information of living organisms is encoded as sequences of nucleic acid bases in DNA, and is protected by the thermodynamically stable secondary structure of the Watson-Crick double helix. The processing and manipulation of gene sequences by macromolecular machines requires that stable segments of duplex DNA be disrupted, and that single-stranded (ss) DNA templates be transiently exposed to the binding sites of DNA associating proteins within the cellular environment. Here we elucidate some of the defining features that control the stability and dynamics of ssDNA secondary structure, using time-resolved methods to detect the presence of transient unstable conformations. Understanding the nature of these instabilities is central to elucidating the mechanisms by which ssDNA templates facilitate protein binding and function.

biophysics↗