Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Abstract

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.

Fig 1

A. Antisense oligonucleotide-mediated depletion of NEIL2 in Chinese hamster V79 cells. Total RNA was isolated from cells treated with antisense or control oligonucleotide or from non-treated cells (NT), and NEIL2 mRNA levels were measured by Q-RTPCR. A.U., arbitrary units. B. Increased mutation frequency at the hprt locus in V79 cells, either non-treated (NT) or treated with NEIL2 control or antisense oligo. The bar graphs represent the means ± standard error from 4 independent experiments.

Fig 2

A. A 5’ ³²P-labeled 51-mer oligo (5-OHU.B11, 1 pmol), was used for DNA glycosylase/AP lyase assay [28] with purified (0.2 pmol) WT (lane 2) or R257L (lane 3) or R103Q (lane 4) NEIL2. Lane 1, no protein. Quantitation of the radioactive bands (lanes 2-4) is represented in a histogram (bottom), with lane 2 arbitrarily set as 1. B. Reconstitution of BER. Complete repair of AP site-containing duplex oligo (10 pmol, top) was measured by incorporation of α³²P-dCMP using purified (0.25 pmol) WT or the NEIL2 variants and other BER proteins (50 fmol each) as indicated. Lane 1 = control, non-damaged oligo. C. BER using NEIL2-FLAG IP. Complete repair of AP site-containing duplex oligo (10 pmol, top) was measured by incorporation of α³²P-dCMP using a FLAG-pulldown complex (0.5μg) of WT (lane 1), R257L (lane 2) or R257L supplemented with a mix of purified PNKP, Pol β, Lig IIIα and XRCC1 (lane 3, 50 fmol of each). Lane 4, mix only.

Fig 3

A. Representative Western analysis showing the levels of FLAG and association of other proteins in the eluted complexes (WT vs. R257L variant). B. Detection of WT vs. variant NEIL2-FLAG (mouse Ab) interaction with RNAP II /Lig IIIα/PNKP/Polβ (rabbit Ab) in cultured cells by Proximity Ligation Assay. WT or variant NEIL2 (mouse Ab) with IgG (rabbit Ab), as controls.

Fig 4

A. NEIL2 transcript levels in control vs. 3’ UTR-specific siRNA-treated cells, quantitated by qPCR. B. Long-range qPCR was used to evaluate genomic DNA damage levels in the WT- and the R257L-expressing cells. Representative gel showing PCR-amplified fragments of HPRT and POLB genes. Amplification of the large fragment was normalized to the amplification product of a small fragment of the corresponding gene. Quantitation of the amplified products is represented in a histogram with the WT arbitrarily set as 100. Upper panel, HEK 293; lower panel, BEAS-2B cells. Other details are described in the Materials and Methods section.