The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding

Nicolas Tarbouriech,Corinne Ducournau,Darren J Hart,Philippe J Mas,Stephanie Hutin,Wim P Burmeister,Petr Man,Christophe N Peyrefitte,Eric Forest,Frédéric Iseni

doi:10.1038/s41467-017-01542-z

Abstract

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta–DNA complex.

Highlights

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell
Poxviruses produce a number of non-essential enzymes involved in DNA precursor metabolism as well as essential proteins that are located at the replication fork
A previous sequence analysis of E9 allowed the delimitation of “poxvirus-specific” inserts[11] that we can redefine based on flexible structural alignments with other family B polymerases

Summary

Introduction

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. For vaccinia virus (VACV), the prototype and most-studied member of the Poxviridae family, the essential replication proteins include: E9, the catalytic subunit of the DNA polymerase[2]; D4, a uracil-DNA glycosylase[3], which together with A20 forms the heterodimeric processivity factor[4]; D5, a hexameric nucleoside triphosphatase[5,6], which contains a superfamily III helicase domain[7] and shows primase activity[8] and I3, a single-stranded DNA-binding protein[9]. High-resolution structures of the D4/A20 interface (D4/A201–50) and of D4/A201–50 bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of an uracil base were obtained[13,14] These data further extend our knowledge on the processivity factor assembly and how DNA synthesis and base excision repair are coupled.

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