Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme

Jiangyu Yan,Jonathan C Trinidad,Tamzin Gristwood,Stephen D Bell,Sonja V Albers,Kelly Schermerhorn,Pietro Roversi,Andrew F Gardner,Nicola G A Abrescia,Thomas R Beattie,Adriana L Rojas

doi:10.1038/ncomms15075

Abstract

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

Highlights

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes
We reconstitute the holoenzyme in vitro, confirm that it exists in vivo, map the interactions of PBP1 and PBP2 with the catalytic PolB1 subunit, elucidate the structure of the small subunits and the holoenzyme and determine the biochemical consequences of small subunit association on the behaviour of PolB1
Our analyses reveal that multi-subunit family B replicative polymerases are not restricted to eukaryotes and further underscore the relationship between the Sulfolobus replication machinery and that of eukaryotes

Summary

Introduction

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. The Crenarchaea, which lack a family D polymerase, instead possess a signature B-family polymerase, named PolB1, that appears to be the main replicative DNA polymerase This enzyme was first purified from Sulfolobus acidocaldarius (Sac) in 1985 Analysis of the purified enzyme led to the conclusion that it was a single-subunit enzyme with a mass of B100 kDa. Subsequent work in Sulfolobus solfataricus (Sso) led to the cloning of gene for the catalytic subunit, revealing it to be a family B DNA polymerase[18]. Purification of the enzyme from Sulfolobus reveals, in contrast to the 30-year old belief, the enzyme is not a single-subunit entity in vivo Rather, it exists as a stable heterotrimer in which the apo-enzyme PolB1 associates with two subunits that we term PolB1-binding protein (PBP) 1 and PBP2. Our analyses reveal that multi-subunit family B replicative polymerases are not restricted to eukaryotes and further underscore the relationship between the Sulfolobus replication machinery and that of eukaryotes

Methods

Results

Conclusion