change in human life expectancy in industrialised coun- tries. Human life expectancy now exceeds 80 years, and it is likely that an increasing number of human beings will reach the maximal lifespan for humans, estimated to be about 120 years, in the 21st century [1]. However, ageing itself, which has been defined as a progressive decline in the physiological and biochemical perfor- mance of cells, tissues and organs that leads to the in- ability of the organism to maintain homeostasis at the molecular, cellular, tissue and organismic level, remains a complex and poorly understood biological process [for review see 2]. Many theories have been advanced to ac- count for the ageing process and none are generally ac- cepted [3,4]. However, the “free radical theory of age- ing”, first proposed by Harman [5], defines ageing as the result of the imperfect protection of the major cellu- lar components (lipids, proteins, DNA) against reactive oxygen species (ROS) generated through aerobic metab- olism. Ever since, many data have accumulated to sup- port this theory [6,7], although other variables, such as genetic factors [8,9], are well known to have an impact on ageing too. ROS could contribute to the ageing pro- cess in a number of ways, since these highly reactive compounds can target lipids, proteins or DNA. A signif- icant body of experimental evidence demonstrates that accumulation of DNA damage as well as the induction of genomic instability are associated with ageing and age-related diseases [10, 11,12]. On the other hand, a large number of physiological protection and repair sys- tems are known to play important roles in maintaining cellular and molecular integrity, by counteracting the ac- cumulation of macromolecular damage and the atten- dant loss of macromolecular functions. For example, the potentially catastrophic accumulation of DNA damage can be antagonised by cellular DNA repair activities. DNA excision repair, a complex biochemical phenome- non directly responsible for the elimination of much DNA damage inflicted by exogenous as well as endoge- nous agents, has been implicated in longevity [13]. The “disposable soma theory” predicts that ageing is caused by an accumulation of somatic damage (arising through a variety of molecular mechanisms, such as stochastic hits endogenously produced by ROS or by environmen- tal factors; or biosynthetic errors in macromolecules) in the presence of less-than-perfect cell maintenance and repair systems whose efficiency is determined genetical- ly and is subject to evolutionary control [14]. Human beings with proven longevity (i.e. centenarians), who often maintain a remarkably good status of general physical health until very late in life and who have es-

The involvement of poly(ADP-ribosyl)ation specifi- cally in DNA base-excision repair has recently been doc- umented [24, 25,26]. In addition to its role in DNA re- pair, poly(ADP-ribosyl)ation has been proposed to be in- volved in DNA replication, gene transcription, cell dif- ferentiation, integration of foreign DNA into the cell ge- nome, suppression of homologous recombination, as well as necrotic or apoptotic cell death [27].

Furthermore, Grube and Bürkle have established a positive correlation between the poly(ADP-ribosyl)ation capacity of peripheral blood mononuclear cells from 13 mammalian species and maximal species-specific life- span, suggesting a possible role for poly(ADP-ribosyl)at- ion as a longevity-assurance factor [28]. Intriguingly, it was reported very recently that telomeres in cells from PARP-1 knockout mice are significantly shorter than those of wildtype mice [29], which may imply that PARP-1 acts to delay the onset of replicative senescence.

Recently we undertook an analysis of the relation tween longevity and poly(ADP-ribosyl)ation in [15]. We showed that maximal table PARP activity was significantly higher in bilised Epstein-Barr virus (EBV)-immortalised blastoid cell lines from of a French population of narians than in cell lines derived from control aged 20–70 years. Interestingly, specific PARP (i.e. total PARP activity corrected for the cellular of immunoreactive PARP-1 protein) was an even er parameter to distinguish between centarians and trols. In this work, we studied whether the difference served in specific PARP activity between and controls might be due to qualitative differences the level of the protein sequence, mediated by in the PARP-1 coding sequence.

ture specific for each primer (see Table 1); followed by 60°C for 4 min. Sequencing reaction products were treated with shrimp al- kaline phosphatase (Amersham) (1 U in a final volume of 20 µl) for 30 min at 37°C. DNA was precipitated in the presence of 0.4 mM MgCl and 2.5 volumes of ethanol (100%) for 15 min at 2 4°C. The pellet was resuspended in 3 µl of loading buffer (form- amide 83%, EDTA 4 mM, Dextran blue 8 mg/ml). Sequencing re- action products were loaded on 4.25% polyacrylamide/6 M urea gels, and analysed on an Applied Biosystems Gene Scanner 377A for 7 h (1.5 kV, 51°C). Signals were collected by the sequence analysis software (version 3.0, Applied Biosystems) and analysed with Sequence Navigator (Applied Biosystems).

Materials and methods

Subjects and DNA samples