Search

1471-0072 (Print) Journal Article Review; The addition to proteins of the negatively charged polymer of ADP-ribose (PAR), which is synthesized by PAR polymerases (PARPs) from NAD(+), is a unique post-translational modification. It regulates not only cell survival and cell-death programmes, but also an increasing number of other biological functions with which novel members of the PARP family have been associated. These functions include transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism.

International audience; In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end. DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.

Oxidative DNA base damage is mainly corrected by the base excision repair (BER) pathway, which can be divided into two subpathways depending on the length of the resynthetized patch, either one nucleotide for short patch BER or several nucleotides for long patch BER. The role of proteins in the course of BER processes has been investigated in vitro using purified enzymes and cell-free extracts. In this study, we have investigated the repair of 8-oxo-7,8-dihydroguanine (8-oxoG) in vivo using wild-type, polymerase beta(-/-) (Polbeta(-/-)), poly(ADP-ribose) polymerase-1(-/-) (PARP-1(-/-)), and Polbeta(-/-)PARP-1(-/-) 3T3 cell lines. We used non replicating plasmids containing a 8-oxoG:C base pair to study the repair of the lesion located in a transcribed sequence (TS) or in a non-transcribed sequence (NTS). The results show that 8-oxoG repair in TS is not significantly impaired in cells deficient in Polbeta or PARP-1 or both. Whereas 8-oxoG repair in NTS is normal in Polbeta-null cells, it is delayed in PARP-1-null cells and greatly impaired in cells deficient in both Polbeta and PARP-1. The removal of 8-oxoG and presumably the cleavage at the resulting apurinic/apyrimidinic site are not affected in the PARP-1(-/-)Polbeta(-/-) cell lines. However, 8-oxoG repair is incomplete, yielding plasmid molecules with a nick at the site of the lesion. Therefore, PARP-1(-/-)Polbeta(-/-) cell lines cannot perform 5'-dRP removal and/or DNA repair synthesis. Furthermore, the poly(ADP-ribosyl)ation activity of PARP-1 is essential for 8-oxoG repair in a Polbeta(-/-) context, because expression of the catalytically inactive PARP-1 (E988K) mutant does not restore 8-oxoG repair, whereas an wild type PARP-1 does.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.

Cell survival after genotoxic stress is determined by a counterbalance of pro- and anti-death factors. Sirtuins (SIRTs) are deacetylases that promote cell survival whereas poly(ADP-ribose) polymerases (PARPs) can act both as survival and death inducing factor and the two protein families are strictly dependent on NAD(+) for their activities. Here we report that SIRT1 modulates PARP-1 activity upon DNA damage. Activation of SIRT1 by resveratrol leads to reduced PARP-1 activity and there is a drastic increase in PAR synthesis in sirt1-null cells. The unbalanced regulation of PARP-1 in the absence of SIRT1 results in AIF (apoptosis inducing factor)-mediated cell death. Our findings establish a functional link between the two NAD+-dependent enzyme systems and provide a physiological interpretation for the mechanism of death in cells lacking SIRT1.

0021-9533 (Print) Journal Article; The DNA damage-dependent poly(ADP-ribose) polymerases-1 and -2 (PARP-1 and PARP-2) are survival factors that share overlapping functions in the detection, signaling and repair of DNA strand breaks resulting from genotoxic lesions in mammalian cells. Here we show that PARP-1 and PARP-2 subnuclear distributions partially overlap, with both proteins accumulating within the nucleolus independently of each other. PARP-2 is enriched within the whole nucleolus and partially colocalizes with the nucleolar factor nucleophosmin/B23. We have identified a nuclear localization signal and a nucleolar localization signal within the N-terminal domain of PARP-2. PARP-2, like PARP-1, interacts with B23 through its N-terminal DNA binding domain. This association is constitutive and does not depend on either PARP activity or ribosomal transcription, but is prevented by mutation of the nucleolar localization signal of PARP-2. PARP-1 and PARP-2, together with B23, are delocalized from the nucleolus upon RNA polymerase I inhibition whereas the nucleolar accumulation of all three proteins is only moderately affected upon oxidative or alkylated DNA damage. Finally, we show that murine fibroblasts deficient in PARP-1 or PARP-2 are not affected in the transcription of ribosomal RNAs. Taken together, these results suggest that the biological role of PARP-1 and PARP-2 within the nucleolus relies on functional nucleolar transcription, without any obvious implication of either PARP on this major nucleolar process.

p53 deficiency confers resistance to doxo (doxorubicin), a clinically active and widely used antitumour anthracycline antibiotic. The purpose of the present study was to investigate the reversal mechanism of doxo resistance by the potent PARP [poly(ADP-ribose) polymerase] inhibitor ANI (4-amino-1,8-naphthalimide) in the p53-deficient breast cancer cell lines EVSA-T and MDA-MB-231. The effects of ANI, in comparison with doxo alone, on doxo-induced apoptosis, were investigated in matched pairs of EVSA-T or MDA-MB-231 with or without ANI co-treatment. Doxo elicited PARP activation as determined by Western blotting and immunofluorescence of poly(ADP-ribose), and ANI enhanced the cytotoxic activity of doxo 2.3 times and in a caspase-dependent manner. The long-term cytotoxic effect was studied by a colony-forming assay. Using this assay, ANI also significantly potentiates the long-term cytotoxic effect with respect to treatment with doxo alone. Decrease in mitochondrial potential together with an increase in cytochrome c release, association of Bax with the mitochondria and caspase 3 activation were also observed in the presence of ANI. Therefore PARP inhibition may represent a novel way of selectively targeting p53-deficient breast cancer cells. The underlying mechanism is probably a potentiation of unrepaired DNA damage, shifting from DNA repair to apoptosis due to the effective inhibition of PARP activity.

The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.

Poly(ADP-ribose) polymerase 2 (PARP-2) is a DNA damage-dependent enzyme that belongs to a growing family of enzymes seemingly involved in genome protection. To gain insight into the physiological role of PARP-2 and to investigate mechanisms ofPARP-2 gene regulation, we cloned and characterized the murine PARP-2 gene. The PARP-2 gene consists of 16 exons and 15 introns spanning about 13 kilobase pairs. Interestingly, the PARP-2 gene lies head to head with the gene encoding the mouse RNase P RNA subunit. The distance between the transcription start sites of the PARP-2 and RNase P RNA genes is 114 base pairs. This suggested that regulation of the expression of both genes may be coordinated through a bi-directional promoter. The PARP-2/RNase P RNA gene organization is conserved in the human. To our knowledge, this is the first report of a RNA polymerase II gene and an RNA polymerase III gene sharing the same promoter region and potentially the same transcriptional control elements. Reporter gene constructs showed that the 113-base pair intergenic region was indeed sufficient for the expression of both genes and revealed the importance of both the TATA and the DSE/Oct-1 expression control elements for the PARP-2 gene transcription. The expression of both genes is clearly independently regulated. PARP-2 is expressed only in certain tissues, and RNase P RNA is expressed in all tissues. This suggests that both genes may be subjected to multiple levels of control and may be regulated by different factors in different cellular contexts.

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T(2)AG(3) repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2(-/-) primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T(2)AG(3) repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity.