The Human Werner Syndrome Protein Stimulates Repair of Oxidative DNA Base Damage by the DNA Glycosylase NEIL1

Sankar Mitra,Istvan Boldogh,Tapas K Hazra,Muralidhar L Hegde,Vilhelm A Bohr,Jeanine A Harrigan,Nadja De Souza Pinto,Jae Wan Lee,Aditi Das,Jason Piotrowski,Marc M Greenberg,William Ramos

doi:10.1074/jbc.m703343200

Abstract

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.

Highlights

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation
In case of LP-base excision repair (BER), ϳ2– 6 nucleotides are incorporated by pol ␤ or pol ␦/⑀, and the displaced 5Ј-flap is cleaved by flap endonuclease 1 (FEN1) prior to strand ligation [11, 12]
The intensity of Raman scattering was recorded DNA Strand Incision Assay for NEIL1—NEIL1 substrates (NEIL1p78 contains no tryptophan residues) for the titration by used were 51-mer oligonucleotides with the damaged base (5-OHU or 8-oxo-G) at residue 26 tion of NEIL1p78 to buffer in a similar manner was served as and 36-mer oligonucleotides, with blank

Summary

EXPERIMENTAL PROCEDURES

Cell Cultures and Knockdown Assays—The human colorectal carcinoma line HCT116 (expressing wild-type p53), a generous gift of Dr B. GlutathioneSepharose beads (50 ␮l, 50% v/v), alone or bound to 1 ml of GST-tagged WRN polypeptides, were incubated for 2 h at 4 °C with 100 ␮g of HCT116 nuclear extract prepared as described previously [39] or purified recombinant NEIL1 (0.1 ␮g) in 250 ␮l of buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 10% glycerol). The intensity of Raman scattering was recorded DNA Strand Incision Assay for NEIL1—NEIL1 substrates (NEIL1p78 contains no tryptophan residues) for the titration by used were 51-mer oligonucleotides (purchased from Midland adding NEIL1 peptide to the HlcRQC solution, where addi- Co.) with the damaged base (5-OHU or 8-oxo-G) at residue 26 tion of NEIL1p78 to buffer in a similar manner was served as (indicated by X, Table 1) and 36-mer oligonucleotides, with blank. The interaction domain of NEIL1 spanning residues 288 to 349 could be mapped using the C termi-

RESULTS

Findings

DISCUSSION