However, one should keep in mind that in the present work we have focused on seeking evidence for changes in the PARP-1 coding region that would confer increased specific activity in centenarians. Our findings might re- flect the “tip of the iceberg”, since (1) additional rare polymorphisms might exist in the coding region, and (2) additional polymorphisms might exist in the non-coding region as well – for instance, in enhancer sequences far upstream of the coding sequences. Those may have an impact on the regulation of gene expression, and, hypo- thetically, changes in PARP-1 gene expression might in- fluence the rate of PARP-1 protein turnover, and thus the functional status of PARP-1 protein.

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 - helical domain of the catalytic fragment of PARP-1. As depicted in Fig. 5, (G. de Murcia, personal communica- tion), the closest neighbour of residue 762 is G888. This amino acid is part of the PARP-1 signature (active site) and is totally conserved during evolution. The distance

polymorphisms have to be considered that could account for the differences observed in poly(ADP-ribosyl)ation capacity between centenarian and control cells. Recently, the in vitro modulation of PARP-1 activity by protein partners has been described. Caldecott et al. [42], and more recently Masson et al. [25], identified PARP-1 as the third partner (in addition to DNA ligase III and DNA β polymerase ) of the base excision repair protein, X-ray repair cross complementing 1 (XRCC1). The interaction between PARP-1 and XRCC1 resulted in the inhibition of the catalytic activity of PARP-1 as well as of its auto- modification level, without modifying the level of pro- tein expression or PARP-1 nick sensor function [25]. On the other hand, the transcription factor Yin Yang 1 (YY1) has been reported to act as a positive regulator of PARP-1 activity in vitro [43].

Finally, the description of several novel PARP homo- logues in mammalians has highlighted the possibility of functional redundancies in the poly(ADP-ribosyl)ation system [19, 20,21]. At least one of the new PARP homo- logues (i.e. PARP-2 [21]) synthesises poly(ADP-ribose) in a DNA-dependent manner. Hence, the global PARP

activity measured in our assay system might in fact re- flect the presence of at least two enzymatic activities (PARP-1 and PARP-2).

Shieh WM, Ame JC, Wilson MV, Wang ZQ, Koh Jacobson MK, Jacobson EL (

) Poly(ADP-ribose) merase null mouse cells synthesize ADP-ribose polymers. Biol Chem

:

–

Smith S, Giriat I, Schmitt A, de Lange T (

) Tankyrase, poly(ADP-ribose) polymerase at human telomeres.

:

–

Amé JC, Rolli V, Schreiber V, Niedergang C, Apiou F, P, Muller S, Höger T, Ménissier-de Murcia J, de Murcia (

) PARP-

, a novel mammalian DNA Poly(ADP-ribose) polymerase. J Biol Chem Bürkle A, Chen G, Küpper J-H, Grube K, Zeller WJ Increased poly(ADP-ribosyl)ation in intact cells by treatment. Carcinogenesis

:

–

Balard B, Giacomoni PU (

) Nicotinamide adenosine nucleotide levels in dimethyl sulfate treated- or mouse epidermis. Mutat Res

:

–

Hjertvik M, Erixon K, Ahnström G (

) Repair of damage in mammalian cells after treatment with UV and methyl sulphate: dicrimination between nucleotide and excision repair by their temperature dependence. Mutat

:

–

Masson M, Niedergang C, Schreiber V, Muller S, Menissier Murcia J, de Murcia G (

) XRCC

is specifically ed with poly(ADP-ribose) polymerase and negatively lates its activity following DNA damage. Mol Cell

:

–

Dantzer F, Schreiber V, Niedergang C, Trucco C, Flatter E, de Rubia G, Oliver J, Rolli V, Ménissier-de Murcia J, de Murcia (

) Involvement of poly(ADP-ribose) polymerase in excision repair. Biochimie

:

–

Bürkle A (

) Poly(ADP-ribose) polymerase and Exp Gerontol

:

–

Grube K, Bürkle A (

) Poly(ADP-ribose) polymerase tivity in mononuclear leucocytes of

mammalian correlates with species-specific life span. Proc Natl Acad USA

:

–

d’Adda di Fagagna F, Hande MP, Tong WM, Lansdorp Wang ZQ, Jackson SP (

) Functions of polymerase in controlling telomere length and stability. Nat Genet

:

–

Sambrook J, Fritsch EF, Maniatis T (

) Molecular – a laboratory manual,

nd edn. Cold Spring Harbor ry Press, New York Baumgartner M, Schneider R, Auer B, Herzog H, M, Hirsch-Kauffmann M (

) Fluorescent in situ of the human nuclear NAD+ ADP-ribosyltransferase (ADPRT) and two secondary sites to human bands

q

,

q

and

q

Cytogenet Cell Genet

Cherney BW, M