Evaluation of the Role of the Vaccinia Virus Uracil DNA Glycosylase and A20 Proteins as Intrinsic Components of the DNA Polymerase Holoenzyme

Kathleen A Boyle,Eleni S Stanitsa,Matthew D Greseth,Jill K Lindgren,Paula Traktman

doi:10.1074/jbc.m111.222216

Kathleen A Boyle, Eleni S Stanitsa + Show 3 more

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https://doi.org/10.1074/jbc.m111.222216

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Abstract

The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ∼1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N' terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.

Highlights

Tide precursor synthesis and metabolism as well as the core set of enzymes and DNA binding proteins that act directly at the replication fork [1, 2]
We address whether the moving polymerase holoenzyme can both incorporate UTP and excise the uracil moiety and whether the polymerase can perform translesion synthesis when it encounters either a dUMP residue or abasic site in the template strand
We demonstrated that the Km value for dUTP of the polymerase is comparable with the Km value for dTTP, the data presented in Fig. 5C suggest that the rate of synthesis is affected by the presence of dUTP in the reaction mixture

Summary

Introduction

Tide precursor synthesis and metabolism as well as the core set of enzymes and DNA binding proteins that act directly at the replication fork [1, 2]. Genetic, genomic, and biochemical analysis has revealed that eight proteins are responsible for vaccinia virus DNA synthesis and maturation. This repertoire includes the catalytic DNA polymerase (E9 [3,4,5,6,7,8,9,10,11]), a stoichiometric component of the heterodimeric processivity factor (A20 [12,13,14,15]), a second component of the processivity factor (D4) that possesses uracil DNA glycosylase (UDG) activity (16 –18), a putative superfamily III helicase with known NTPase and DNA primase activity (D5 (19 –23)), a serine/threonine protein kinase (B1 (24 –26)), an abundant phosphoprotein with essential roles in viral replication, transcription, and morphogenesis (H5 [27]), a single-strand DNA-binding protein (I3 [28, 29]3), and a DNA ligase (A50 [30]). The virally encoded proteins involved in nucleotide precursor synthesis and metabolism have been reviewed elsewhere [33, 34]

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