Expression of a germline variant in the N-terminal domain of the human DNA glycosylase NTHL1 induces cellular transformation without impairing enzymatic function or substrate specificity.

Carolyn G Marsden,Miral Dizdaroglu,Robyn L Maher,Joann B Sweasy,Pawel Jaruga,David S Pederson,Erdem Coskun

doi:10.18632/oncotarget.27548

Abstract

Oxidatively-induced DNA damage, widely accepted as a key player in the onset of cancer, is predominantly repaired by base excision repair (BER). BER is initiated by DNA glycosylases, which locate and remove damaged bases from DNA. NTHL1 is a bifunctional DNA glycosylase in mammalian cells that predominantly removes oxidized pyrimidines. In this study, we investigated a germline variant in the N-terminal domain of NTHL1, R33K. Expression of NTHL1 R33K in human MCF10A cells resulted in increased proliferation and anchorage-independent growth compared to NTHL1 WT-expressing cells. However, wt-NTHL1 and R33K-NTHL1 exhibited similar substrate specificity, excision kinetics, and enzyme turnover in vitro and in vivo. The results of this study indicate an important function of R33 in BER that is disrupted by the R33K mutation. Furthermore, the cellular transformation induced by R33K-NTHL1 expression suggests that humans harboring this germline variant may be at increased risk for cancer incidence.

Highlights

Maintenance of genomic integrity requires proper functioning of the base excision repair (BER) pathway, which is responsible for the repair of at least 30,000 lesions per cell per day arising from endogenous insults [1, 2]
As a human germline mutation, we were first interested in testing whether R33K-NTHL1 expression in MCF10A cells could induce phenotypes indicative of cellular transformation
To achieve knockdown of endogenous NTHL1 without impacting expression of exogenously expressed wt-NTHL1 or R33KNTHL1, two silent mutations were generated in the open reading frame (ORF) of wt-NTHL1 and R33K-NTHL1 within the sequence targeted by two overlapping short-hairpin RNAs (shRNA) (Figure 2B)

Summary

Introduction

Maintenance of genomic integrity requires proper functioning of the base excision repair (BER) pathway, which is responsible for the repair of at least 30,000 lesions per cell per day arising from endogenous insults [1, 2]. Bifunctional DNA glycosylases, which generally repair oxidatively-induced DNA lesions, possess both glycosylase and lyase activity. Bifunctional DNA glycosylases can potentially cleave the DNA backbone (utilizing their lyase activity) generating DNA ends that are further processed by AP endonuclease I (APE1) or polynucleotide kinase (PNK). Consisting of only four-five reaction steps, BER has traditionally been considered one of the simplest DNA repair pathways; recent studies have begun to unveil the complexities of BER [2]. BER can be reconstituted in vitro by combining the enzymes necessary for each step in the pathway, in vivo studies have begun to unveil the complex interplay between BER enzymes and other repair and non-repair proteins driving accurate and efficient repair in the cellular environment

Methods

Results

Discussion

Conclusion