The θ Subunit of Escherichia coli DNA Polymerase III: a Role in Stabilizing the ε Proofreading Subunit

Abstract

ABSTRACT The function of the θ subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. θ is a tightly bound component of the DNA polymerase III core, which contains the α subunit (polymerase), the ε subunit (3′→5′ exonuclease), and the θ subunit, in the linear order α-ε-θ. Previous studies have shown that the θ subunit is not essential, as strains carrying a deletion of the holE gene (which encodes θ) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3′-exonuclease activity of ε to be modestly enhanced by the presence of θ. Here, we report a series of genetic experiments that suggest that θ has a stabilizing role for the ε proofreading subunit. The observations include (i) defined Δ holE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong Δ holE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of α-ε interactions by the presence of θ. θ appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of θ might be uniquely beneficial in those instances where the proofreading 3′-exonuclease is not part of the polymerase polypeptide.