DNA Polymerase λ Protects Mouse Fibroblasts against Oxidative DNA Damage and Is Recruited to Sites of DNA Damage/Repair

Elena K Braithwaite,Padmini S Kedar,Li Lan,Yaroslava Y Polosina,Kenjiro Asagoshi,Vladimir P Poltoratsky,Julie K Horton,Holly Miller,George W Teebor,Akira Yasui,Samuel H Wilson

doi:10.1074/jbc.c500256200

Abstract

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

Highlights

Mammalian cells express a large number of DNA polymerases that are considered to differ from one another according to specialized roles in DNA replication, repair, and other essential DNA transactions such as somatic hypermutation [1]
Mouse embryonic fibroblast (MEF) cells that are deficient in pol ␤ are hypersensitive to the cytotoxic effects of DNA alkylating agents [6], and this appears to be due to the persistence of toxic intermediates of DNA repair that are normally removed by pol ␤ in wild-type cells [7]
DNA Polymerase ␭Ϫ/Ϫ Cells Are Hypersensitive to Oxidative DNA Damage-causing Agents—mouse embryonic fibroblast (MEF) that are pol ␭-deficient by virtue of homozygous gene deletion were examined for sensitivity to DNA damaging agents

Summary

Introduction

Mammalian cells express a large number of DNA polymerases that are considered to differ from one another according to specialized roles in DNA replication, repair, and other essential DNA transactions such as somatic hypermutation [1]. In vitro BER time course reactions were performed by incubating this 35-base pair oligonucleotide duplex (250 nM) with 15 ␮g of MEF cell extracts at 37 °C in a buffer containing 25 mM Tris, pH 7, 60 mM NaCl, 2 mM dithiothreitol, 0.2 mM EDTA, 1 mg/ml bovine serum albumin, and 10% (v/v) glycerol.

Results

Conclusion