The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Leena Maddukuri,Sarah Eddy,Robert L Eoff,Amit Ketkar,Maroof K Zafar

doi:10.1093/nar/gku913

Leena Maddukuri, Sarah Eddy + Show 3 more

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https://doi.org/10.1093/nar/gku913

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Abstract

Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.

Highlights

DNA damage is a barrier to faithful replication of the genome
translesion DNA synthesis (TLS) is an important mechanism for tolerating DNA damage and Y-family pols are central to the bypass of many DNA adducts [5,6,7]
We find that the rate constant for full-length extension by hpol ␬ on unmodified DNA is increased in the presence of Werner syndrome protein (WRN) and that this stimulation can be mostly attributed to the RecQ C-terminal (RQC) domain

Summary

Introduction

DNA damage is a barrier to faithful replication of the genome. Biological systems are continuously exposed to agents that react with DNA and alter its physicochemical properties, which can impair normal nucleic acid metabolism [1,2]. DNA adducts that are not repaired can block replication fork progression [3,4]. The activation of replication stress response pathways can lead to the recruitment of specialized DNA polymerases (pols) to perform translesion synthesis across adducts [5]. The Y-family DNA polymerases are central to this process [6]. Humans have four Y-family members that participate in a number of pathways involved in cellular responses to DNA damage [6]. Each human Y-family pol bears unique structural characteristic and lesion bypass properties [7]. Understanding the distinct mechanism of action for each of the four human Y-family pols is an ongoing area of investigation, as the diversity of form and function found within this family of enzymes is remarkable

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