T4 Replication Accessory Proteins Visualized as Complexes with DNA by Cryoelectron Microscopy

Abstract

Replication of bacteriophage T4 is accomplished by a set of proteins encoded by the T4 genome.Central to replication is the DNA polymerase, the product of gene 43 (g43p); while it contains the intrinsic functions of template-directed DNA synthesis, it requires a set of accessory proteins as a minimal system to function with a rate, fidelity and processivity comparable to those obtainedin vivo.These accessory proteins are g44p, g45p and g62p; g32p, a single-stranded DNA binding protein, also assists in the replication process, bothin vitroandin vivo.In the absence of DNA, g44p and g62p form a 4:1 complex which has a DNA-dependent ATPase activity; g45p is a trimer in solution. Their association state when bound to DNA is however unknown, as is the mechanism by which they dramatically improve the replication activity of g43p.We have examined the complex of the three replication accessory proteins and g32p as a complex with DNA by cryoelectron microscopy, avoiding stains or fixatives which may disrupt the labile structures formed. Under conditions which promote the polymerase-enhancing activity of this four-protein system, distinct "hash mark" structures are visible along strands of replicative form M13 DNA (figure 1). These structures are formed only in the presence of all four proteins and ATP; absence of any one of the proteins or ATP, or substitution of non-hydrolyzable ATP analogues, fails to yield any of the hash marks.