genotype at these two loci (locus T C: total n=40 indi- 2444 viduals; T/T(n=27) versus T/C(n=11), P=0.15; T/T(n=27) versus C/C(n=2), P=0.86; T/C(n=11) versus C/C(n=2), P=0.55. Locus T C: total n=38 individuals; T/T(n=16) 1011 versus T/C(n=18), P=0.81; T/T(n=16) versus C/C(n=4), P=0.63; T/C(n=18) versus C/C(n=4), P=0.86). In conclu- sion, the results of the analysis of PARP activity as a function of observed alleles at nucleotide positions 1,011 and 2,444, did not reveal any significant differences among individuals with different genotypes.

Discussion

In two independent sets of experiments, some of us showed previously that cellular poly(ADP-ribosyl)ation capacity is associated with longevity. First, PARP activi- ty in permeabilised mononuclear blood cells was posi- tively correlated with lifespan in mammalian species [28] and second, in permeabilised EBV-immortalised lymphoblastoid cell lines from human centenarians, PARP activity was significantly higher compared with control adults aged 20–70 [15]. These observations are in line with the hypothesis that a higher poly(ADP-ribo- syl)ation capacity would contribute to a longer lifespan of an organism, by better assuring genomic integrity and stability over chronological time [33].

One should keep in mind that the type of activity say used reflects only the synthesis but not catabolism poly(ADP-ribose) [28], the latter being catalysed by poly(ADP-ribose) glycohydrolase. Moreover, in

1 cDNA of 18 individual samples gave clear proof for the presence of these polymorphisms. While the small size of the sample analysed did not allow us to interpret data as a serious association study with any kind of phe- notype, we nevertheless observed that the genotypes cor- responding to the transitions C T, G A and T C 402 1215 2444 were in total linkage disequilibrium. In contrast, the fourth transition, T C, showed genotype combinations 1011 totally independent of the previous one, suggesting two independent ancestral events. This observation made it highly interesting to analyse the two polymorphisms T C and T C in a larger population. The former, as 2444 1011 it leads to the V762A exchange, had to be compared with the PARP activity values we had already measured in the previous study. The latter, T C, even if silent at 1011 the protein sequence level, represents a potentially useful molecular tool suitable for any association or linkage analysis between the PARP-1 gene and a given pheno- type. In order to study these two sequence polymor- phisms in a larger population, it was necessary to devel- op an approach allowing us to study genomic DNA rath- er than mRNA extracted from cell lines. Therefore, we designed an allele-specific amplification method in con- junction with a fluorescent read-out system, which al- lowed us to analyse much larger numbers of samples. By using this reproducible and convenient method, we ob- tained data from 648 samples (324 centenarians and 324 controls). We calculated allele and genotype fre- quencies in each population in order to compare them, and analysed the association with human longevity. When we compared allelic frequencies and genotypes from the two populations studied, we found a clear ab- sence of genetic association with human longevity both at the T C and the T C locus. We even observed a 2444 1011 perfect identity of allele-frequencies between the two populations for the T C locus (Fig. 3). On the other 2444 hand, we expected that T C transition, which is re- 2444 sponsible for a V762A exchange in the catalytic domain of the enzyme, would influence PARP activity. Although the structural consequence of a valine-to-alanine transi- tion is expected to be rather weak, as it leads to the loss of a single methyl residue on the amino acid lateral chain, such an exchange has already been shown to mod- ify the thermo-sensitivity and finally the activity of a protein such as the methylenetetrahydrofolate reductase (MTHFR) [38]. Moreover the C-terminus region, in which V762A is located, is the region most strictly con- served during evolution of the PARP-1 protein sequence. This segment can be separated from the rest of the pro- tein, generating a 40 kDa C-terminal fragment that re- tains a basal level of catalytic activity [39]. The crystal structure of the catalytic fragment of chicken PARP-1 α has been described [40]. Residue 762 is located in the -

Hence, in order to relate a specific genotype pattern to a high or low enzyme activity, we focused on genotype distributions from individuals who had previously been tested for PARP activity. However, none of the genotype combinations, at any locus studied, showed any associa- tion either with a particularly high or low maximal PARP activity (expressed in cpm/106), or with the specific en- zyme activity.

Finally, analysis of PARP activity as a function of the observed alleles at nucleotide positions 1,011 and 2,444 of PARP-1 cDNA did not reveal any significant differ- ence between individuals with different genotypes. This suggests that the polymorphisms T C and T C do 1011 2444 not have any impact on the activity of the enzyme. Like- wise, in independent work, it was recently shown that putative differences in enzyme kinetic parameters of PARP-1, such as V and K , could not account for the max m differences observed in the poly(ADP-ribosyl)ation ca- pacity of human and rat cells [41], although the latter was positively correlated with lifespan of these mamma- lian species [28].