Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation

Abstract

Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N-glycosylases. One of these, n th endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and subSaharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.DNA repair | mutagenesis | MCF10A breast epithelial cells T he base excision repair (BER) pathway is responsible for the removal of at least 20,000 DNA lesions per cell per day (1), making it critical for the maintenance of genomic stability. The simplest and most common form of BER is short patch BER, which can be initiated by one of several different DNA glycosylases, each having preferences for specific types of lesions (2). Monofunctional DNA glycosylases recognize DNA lesions and catalyze the hydrolysis of the N-glycosylic bond to generate an abasic site. The abasic site is nicked at its 5′ side by the APE1 endonuclease, leaving a 3′OH and a 5′deoxyribose phosphate (dRP). DNA polymerase beta (pol β) fills in the single nucleotide gap and catalyzes removal of the dRP group. Bifunctional glycosylases, which usually recognize oxidative lesions, generate an abasic site and then catalyze its removal via β-elimination to generate a 3′dRP and 5′phosphate. APE1 then catalyzes removal of the 3′dRP, leaving a 3′OH, to which pol β can bind and fill in the resulting single nucleotide gap. In both cases, the XRCC1/ Ligase IIIα or XRCC1/Ligase I complex catalyzes ligation of the resulting ends. An alternative BER pathway, that does not depend on APE1, is used when the NEIL glycosylases initiate repair (3). NEIL 1, 2, and perhaps 3 catalyze excision of the damaged base via β,δ elimination, leaving a 3′phosphate and a 5′phosphate. The 3′phosphate is removed by polynucleotide kinase, leaving a gap that is most often filled by pol β, followed by ligation.NTH1 is one of four human bifunctio...

The R280H X-ray cross-complementing 1 germline variant induces genomic instability and cellular transformation

“…For H 2 O 2 treatment, plates were incubated with 30 mM H 2 O 2 in serum free media for 30 minutes on ice, then replaced with fresh complete media and allowed to recover for 1, 2, and 6 hours as described (34). For MMS treatment, cells were incubated with 2 mM MMS for 2h and then allowed to recover for 1, 2, and 4 hours (33).…”

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

“…hMPG-Previous reports suggest that reduced activity of DNA glycosylases may promote genomic instability (23). Given that R141Q and R120C showed reduced activity, it is possible that these variants may facilitate an increase in ⑀A-induced mutations compared with WT hMPG.…”

“…Many DNA repair gene polymorphisms have been identified; some have been shown to affect protein function (17)(18)(19)(20) and have been associated with an increased incidence of cancer (21,22). In fact, an SNP variant of hNTH1, another BER DNA glycosylase, showed loss of activity and induced genomic instability in human and mouse cells (23). A number of hMPG nsSNPs were identified in human populations by genetic analysis and validated, but their clinical and functional significance are not known.…”

Multifaceted roles for thymine DNA glycosylase in embryonic development and human carcinogenesis

“…TDG polymorphism is also associated with genomic instability and cellular transformation [67,68]. The D239Y variant occurs in 6.2% of global population.…”

“…The D239Y variant occurs in 6.2% of global population. The D239Y-expressing cells are more sensitive to DNA damage and are more active in DNA damage response [67]. The G199S variant occurs in 10% of global population [68].…”