2.4. Statistical analysis

et al., 2006; http://www.ikmb.uni-kiel.de/software.html). The genetic markers were subjected to allelic (CCA) and genotypic (CCG) case–control comparisons (x2 test, P : P value obtainedfrom anallele-basedcase–controlcomparison, using1degree CCA of freedom (df); P : P value obtained from a genotype-based case–control CCG comparison, usinga x2 test with 2 df). TaggingSNPs ofEXO1andTCF3 were selected based on the HapMap genotypes of Utah residents with ancestry from northern and western Europe (CEU; http://www.hapmap.org/) and the pairwise tagging option implemented in the Haploview v3.32 program (Barrett et al., 2005; http:// www.broad.mit.edu/mpg/haploview/) (minor allele frequency 0.03, pairwise r2   0.8, P > 0.01; for tag SNPs, see Table S3). Haploview was also used for HWE determining the LD measure r2 between SNPs in controls. The seven EXO1 SNPs (Table 2) typed in the German sample and the functionally relevant SNP rs1776180 typedinthe Frenchsetwereanalyzedbylogistic regressionusingan additivemodel

iad“1090 Rea  °3 é e°25 oe2°% °1.5 . e eee1 ce8ee e.0.5e e oe? = ee e e eo+ T —T Try T 1240055000 240065000 240075000 240085000 240095000 240105000 240115000physical position (bases)240125000

vZ00SL YS8L00SLrs!eeLeorys!oseessGYLOLLLS!1gzeLyiss@ri9Lcissovezrorss202 L6EL183.S966rbys4e966rl rsszs66riyss66S46/z781sve6rl yss€v6selsvoosezzsszeeertyss6066rl ys!909 LOx3eeggzszsiz986ri ys)ese6rl ys!sseeriys:sLLU9LLESsLESSEOLSSsBLE9LLES6LLOLLLS)sOBF9LLES2sbBLOZZESLevsveses!‘9662080183eisseoLsse6orszessZ60rSLES)SyrLgsiiss2e6811Z181606g9sz1s)veLvaszisss66z08015)gszzezes!zezteg9s:oseeerorssgeerzszsssrnraerrniSeriesEXO1 NM_130398¢°TAAATG+345 6 7 8 910 1 12 1314 152Fig. 1. (Continued).

by means of the ‘First Exon and Promoter Prediction Program for Human DNA’ (http://rulai.cshl.org/tools/FirstEF/). Sequencing traces were inspected for the presence of SNPs and insertions/deletions using novoSNP (Weckx et al., 2005).

variants of EXO1 inserted into pDB20 vector by applying standard yeast transformation techniques. Three independent clones were generated for each construct. Clones containing empty expression vector served as negative control, yeast strain U687 (Drad51) deficient for rad51 but not for exo1 (Qiu et al., 1999) served as specificity control. Yeast cells were spread on growth plates, exposed to different UV dosages ranging from 0 to 40 mJ/cm2 (UV-B light source; Phillips TL 20/W12, Eindhoven, Netherlands) and incubated at 30 8C for 48 h. The number of resulting colonies was counted and calculated as percentage of untreated controls.

2.6. Construction of plasmids

Plasmids for over-expression of human EXO1 protein in yeast cells were generatedbyinsertingfulllengthEXO1cDNAintoexpressionvectorpDB20(Becker et al., 1991). Constructs for analysis of EXO1 50-UTR were designed by insertion of a 584 bp fragment of the 50-UTR region into vector pGL3control (Promega, Madison, WI). Constructs for analysis of EXO1 50-proximal promoter were designed by inserting 1199 bp of the EXO1 promoter region into vector pGL3Enhancer (Promega).Allele-specificconstructswereestablishedbysite-directedmutagenesis using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). The over- expression construct pMyc-E47-EYFP was kindly provided by Naihe Jing (Shanghai InstitutesforBiologicalSciences,Shanghai,China).ForRNAi-mediatedknock-down of E47/TCF3 expression, two independent target sequences directed against E47/ TCF3mRNAwereinsertedintovectorpSUPER.Neo + GFP(OligoEngine,Seattle,WA).

2.8. Cell culture and transfection

HumanembryonalkidneyHEK293(ACC305),cervicalcarcinomaHeLacells(ACC 57), human acute monocytic leukemia cell line THP-1 (ACC16) and human T cell leukemia Jurkat cells (AC282) were cultured under standard conditions. Transfec- tionswereperformedusingFugene6TM(Roche,Basel,Switzerland)accordingtothe manufacturer’s manual.

2.9. Dual luciferase assay

For quantitative analysis of the 50-UTR and 50-proximal promoter regions, pGL3 constructswereusedfordualluciferasereportergeneassay(Promega)asdescribed before (Till et al., 2005).

The UV-sensitive yeast strain FDER (Dexo1/Drad51) (Qiu et al., 1999) was used for generation of individual clones overexpressing the allele-specific

(Applied Biosystems, Foster City, USA). The investigation of DNA repair genes represented the explorative stage of our study and served to identify markers for further genetic and functional follow-up and replication (Fig. 1A). For the associationanalyses, we applied an established longevity study design (Nebel et al., 2005) by comparing German centenarians (n = 395; aged 100–110 years) to younger controls (n = 411; aged 60–75years) that were matched by ancestry, gender and geographic origin within Germany.

A total of 123 lymphoblastoid cell lines were obtained from heparinized samples of unrelated healthy donors (from the Popgen Biobank) (Krawczak et 2006). DNA was genotyped for rs1776180 using a TaqMan1 SNP Genotyping (Applied Biosystems, Foster City, USA). Total RNA was extracted with the RNeasy Kit according to the manufacturer’s guidelines (Qiagen, Hilden, Real-time PCR was performed using a 7900HT Fast Real Time PCR-System Biosystems, Foster City, USA). Expression levels of EXO1 (assay were calculated with the standard curve method (Livak and Schmittgen, Significance was determined using the Mann–Whitney U-test.