Structure of New Binary and Ternary DNA Polymerase Complexes From Bacteriophage RB69.

Jongseo Park,Hyung-Seop Youn,Jimin Wang,Soo Hyun Eom,Youngjin Lee,Jun Yop An

doi:10.3389/fmolb.2021.704813

Abstract

DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.

Highlights

DNA replication at the replication fork is the semi-discontinuous process; one strand is the leading strand, and the other strand is the lagging strand (Hamdan et al, 2009; Yao and O’Donnell, 2008)
DNA polymerases are very dynamic, undergo many conformational changes with correct incoming dNTPs, and transverse various distinctive functional states such as preinsertion or post-insertion according to their interactions with both dNTPs and the P/T duplex (Franklin et al, 2001; Berman et al, 2007)
The N-terminal domain (NTD) of RB69pol and the part of the P/T duplex away from the active site participate in the dimerization of the two complexes

Summary

INTRODUCTION

DNA replication at the replication fork is the semi-discontinuous process; one strand is the leading strand, and the other strand is the lagging strand (Hamdan et al, 2009; Yao and O’Donnell, 2008). The DNA polymerase from RB69 phage (RB69pol) belongs to the B-family pols and shares a high degree of similarity with human pols (Wang et al, 1997) It contains two activities (polymerase and exonuclease) and is a classic model for studying DNA replication (Wang et al, 1997; Alberts, 2003). The apparent binding affinities for both Watson–Crick base-paired dNTPs and divalent metal ions are very high (apparent Kd ∼50 μM) (Xia et al, 2013a) These apparent values are averaged from many conformational steps. Intermediate structures between the classic open binary complex and the fully closed ternary complex have already been reported for other DNA pols, for example, an “ajar” intermediate (Wu and Beese, 2011) How these intermediates are correlated with the base selectivity of nucleotide incorporation by DNA pols remains poorly understood

MATERIALS AND METHODS

RESULTS AND DISCUSSION

A New Closed Ternary Complex With Single Divalent Metal Ion Bound

DATA AVAILABILITY STATEMENT