T4 gp32 forms dynamic, compacting filaments on ssDNA that reorganize through interprotein interactions.

Abstract

Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication, recombination, and repair. gp32 forms cooperative filaments on ssDNA through interprotein interactions mediated by its N-terminal domain. Detailed understanding of gp32 filament structure and organization remains incomplete, particularly with respect to longer, biologically-relevant DNA lengths. We use optical tweezers and atomic force microscopy (AFM) to probe the structure and binding dynamics of gp32 on long (∼7-8 knt) ssDNA substrates. We find that cooperative binding of gp32 reduces the contour length of ssDNA by ∼40%, suggesting that gp32 filaments helically wind ssDNA, a binding conformation that may be favorable for the synthesis of dsDNA on the ssDNA template during replication. Additionally, gp32 binding gives rise to multiphasic DNA extension changes which reflect competition between increases in ssDNA persistence length and changes in its contour length. Notably, under conditions of high force or high protein concentration, cooperative gp32 clusters are able to modulate their binding footprint to drive elongation of the DNA molecule relative to its compacted state. This binding mode is unstable and marked by an additional phase of rapid protein dissociation not seen at lower concentrations, suggesting a possible mechanism for prompt gp32 dissociation during DNA replication.