Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase.

Heike M Kropp,Kay Diederichs,Andreas Marx,Samra Ludmann,Karin Betz

doi:10.1002/cbic.202100306

Abstract

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B‐family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B‐family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’‐overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B‐family DNA polymerases.

Highlights

Several archaea live at temperatures exceeding 80 °C and since DNA is prone to deamination at elevated temperatures, this results in increased cytosine to uracil and adenine to hypoxanthine conversions (Figure 1 A).[1]
Connolly and colleagues found that archaeal B-family DNA polymerases have evolved a so called “read ahead” mechanism to sense uracil in the template strand, stalling DNA polymerase promoted DNA synthesis of nascent DNA before the deaminated nucleobase reaches the polymerase active site.[4]
Superposing KOD-21nt with the previously solved structure of Thermococcus gorgonarius (Tgo) DNA Polymerase with an uracil-containing template (Tgo-U), we identified that the binding pocket responsible for the recognition of deaminated nucleobases described in the introduction is in close proximity to the + 6 position of the single stranded template overhang when the enzyme is in an active polymerization state

Summary

Introduction

Several archaea live at temperatures exceeding 80 °C and since DNA is prone to deamination at elevated temperatures, this results in increased cytosine to uracil and adenine to hypoxanthine conversions (Figure 1 A).[1] These alterations will lead to G : C to A : T and vice versa transition mutations in the genome and their genomic integrity is at higher risk.[1] All organisms contain repair systems[2] that recognize and excise damaged nucleotides e. G., uracil from DNA by glycosidic bond cleavage that initiates the base excision repair pathway[3] and restores the G : Cbase pair. Connolly and colleagues found that archaeal B-family DNA polymerases have evolved a so called “read ahead” mechanism to sense uracil in the template strand, stalling DNA polymerase promoted DNA synthesis of nascent DNA before the deaminated nucleobase reaches the polymerase active site.[4] Similar results [b] Dr K.

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