Y1 Y7 Y16 Y17 Y31 Y45 Y49 Y113 Y117 Y164 M422 M423 M439 M440 M443 M444 M445 M447 M449 M456 C218 C224 C407 C495 C524 C576 C580 C583 C625 C627

CC genotypes of the Lys751Gln polymorphism are significantly less common in centenarians than in young subjects, while the frequencies of the Arg156Arg and Asp312Asn polymorphisms are not significantly associated with aging. The Lys751Gln polymorphism is located within C-terminal domain (CTD) of XPD, outside of its helicase or ATPase domains. However, both p53 protein and the p44 subunit of the core TFIIH associate with CTD of XPD, modulating XPD’s helicase activity. In addi- tion, XPD stabilizes the quaternary structure of TFIIH. Therefore, the Lys751Gln variants might differentially ‘respond’ to p53 or p44 binding, and/or differentially affect stability of TFIIH (Coin et al. 1998; Schultz et al. 2000). Comparison of the XPD genotypes at codon 751 with DNA repair efficiency showed that carriers of the Lys-encoding A allele at position 751 had a significantly higher number of benzo[a]pyrene diol epoxide-induced DNA adducts, X-ray induced single stranded DNA breaks, double stranded DNA breaks and chromatid aberrations compared to carriers of the Gln-encoding C allele, indicating that the Lys variant is associated with the decreased DNA repair activity of XPD (Lunn et al. 2000; Vodicka et al. 2004; Naccarati et al. 2006; Zhao et al. 2008). In agreement with previous findings, the most chromatid breaks were found in elderly carriers of the Lys/Lys variants of the XPD Lys751Gln polymorphism. This polymorphism was also significantly associated with the level of cells with micronuclei (Kazim´ırova´ et al. 2009). On the other hand, Laine´ and co-workers found that this

Fig. 3 Methylation profile of the XPD promoter fragment analyzed in blood mononuclear cells of young, middle-aged and extremely old human subjects. Genomic DNA isolated from blood mononuclear cells was bisulfite-modified and used as a template for PCR in which the XPD promoter fragment was amplified. The amplified 377 bp fragment was cloned into the pGEM-T vector. Three clones obtained from each person were sequenced. Y (young) 22–37 years old subjects, n = 10; M (middle-aged) 65.3–66.5 years old subjects, n = 10; C (extremely old) 100–102.4 years old subjects, n = 10, TSS transcription start site

XPD promoter in blood mononuclear cells—the only biological material easily obtainable from living donors. To our knowledge, this is the first report describing such age-dependent changes of the XPD in humans. Analysis of the available cases showed no

the Arg156Arg and Asp312Asn polymorphisms are not significantly associated with longevity are con- sistent with such a conclusion.

capacity and on basal transcription (Laine´ et al. 2007). The DNA repair capacity is crucial for vulnerability to cancerogenesis, therefore, the XPD polymorphisms were extensively studied in cancer patients. Results regarding the Lys751Gln polymor- phism are relatively consistent: if any significant positive association with cancer risk is present, it usually is the CC genotype (Gln/Gln) that promotes cancer development. Such unfavorable association was shown, for example, for breast (Justenhoven et al. 2004; Terry et al. 2004), lung (Spitz et al. 2001; Kiyohara and Yoshimazu 2007; Hung et al. 2008), esophagus (Liu et al. 2007; Tse et al. 2008; Wang et al. 2008) cancers, melanoma (Millikan et al. 2006; Li et al. 2006) and acute lymphoblastic leukemia (Wang et al. 2008). Such findings are inconsistent with the functional data described above, that shows either that the Lys751Gln polymorphism does not influence protein function (Laine´ et al. 2007) or that it’s the Lys/Lys, not Gln/Gln phenotype, which is associated with altered DNA repair function of XPD (Lunn et al. 2000; Vodicka et al. 2004; Naccarati et al. 2006; Zhao et al. 2008; Kazim´ırova´ et al. 2009). If it is true, the reason for such a discrepancy is currently unknown. At this stage, our finding is consistent both with in vitro and with clinical findings: the decreased frequencies of function- impairing AA and cancer-prone CC genotypes of the XPD Lys751Gln polymorphism promote longev- ity thanks to efficient DNA repair and lower risk of developing life-threatening disease.

The study has several limitations. First one is the relatively small number of individuals subjected to promoter methylation analysis and that only three clones from each individual were analyzed. Blood mononuclear cell fraction consists of different cell types and, therefore, cell type-specific age-related methylation changes could be missed. Second, the XPD polymorphism frequencies were analyzed only in young and in centenarian age groups, while the results are usually more convincing if additional group of intermediate age is also tested. Unfortu- nately, we were not able to collect group of this age large enough to perform statistical analysis. Third, the study groups subjected to polymorphism analysis differed with respect to sex distribution: while in young controls males/females ratio was 1/0.8, in centenarians it was 1/6.8. However, to our knowl- edge, no sex-related differences in XPD function were ever reported.

To sum up, in this work we show for the first time that the XPD promoter methylation pattern changes with age in human blood mononuclear cells and that the changes are already present at middle age. We also show that the amount of XPD mRNA decreases with progressing age and that the Lys751Gln poly- morphism affecting protein function is associated with aging. Such XPD-related changes support the notion that aging is associated with altered DNA repair and transcription.