DNA-induced dimerization of poly(ADP-ribose) polymerase-1 triggers its activation.

In response to DNA strand breaks in the genome of higher eukaryotes, poly(ADP-ribose)polymerase 1 (PARP-1) catalyses the covalent attachment of ADP-ribose units from NAD(+) to various nuclear acceptor proteins including PARP-1 itself. This post-translational modification affecting proteins involved in chromatin architecture and in DNA repair plays a critical role in cell survival as well as in caspase-independent cell death. Although PARP-1 has been best-studied for its role in genome stability, several recent reports have demonstrated its role in the regulation of transcription. In this study, fluorescence spectroscopy and biochemical techniques are used to investigate the association of the amino-terminal DNA-binding domain of human PARP-1 (hPARP-1 DBD) with various DNA substrates, characterized by different DNA ends and sequence features (5'- or 3'-recessed end, double strands, telomeric repeats, and the palindromic sequence of a Not I restriction site). The correlation between the binding mode of hPARP-1 DBD to the DNA oligoduplexes and the enzymatic activation of hPARP-1 is analyzed. We show that hPARP-1 DBD binds a 5'-recessed DNA end cooperatively with a stoichiometry of two proteins per DNA molecule. In contrast, a 1:1 stoichiometry is found in the presence of a 3'-recessed end and double-strand DNA. A palindromic structure like the Not I restriction site is shown to induce protein dimerization and high enzymatic activation, suggesting that it can represent a recognition element for hPARP-1 in undamaged cells. Protein dimerization is found to be a requisite for high enzymatic activity. Taken together, our data allow further characterization of the features of hPARP-1 recognition in damaged cells and bring additional evidence that hPARP-1 may also play a role in undamaged cells.

Reduction in colorectal cancer mortality by fecal occult blood screening in a French controlled study.

BACKGROUND & AIMS: Several randomized population-based studies have shown that screening for colorectal cancer (CRC) by fecal occult blood tests (FOBTs) can reduce CRC mortality. The aim of this French population-based study was to assess whether a similar benefit could be obtained in countries characterized by high performances in the diagnosis and management of CRC. METHODS: Small-sized geographic areas, including 91,199 individuals aged 45-74 years, were allocated to either FOBT screening or no screening. Six screening rounds were performed. The FOBT was performed without diet restriction and was sent to a central analysis center and processed without rehydration. Screening group participants who had a positive test result were offered a full colonoscopy. The entire population was followed up for 11 years after study entry. RESULTS: Acceptability of the test was 52.8% at the first screening round and varied between 53.8% and 58.3% in the successive rounds. Positivity rates were 2.1% initially and 1.4% on average in the successive rounds. CRC mortality was significantly lower in the screening population compared with the control population (mortality ratio, 0.84; 95% confidence interval, 0.71-0.99). The reduction in CRC mortality was more pronounced in those who participated at least once (mortality ratio, 0.67; 95% confidence interval, 0.56-0.81). CONCLUSIONS: Our findings, together with the results of other trials, suggest that biennial screening by FOBTs can reduce CRC mortality regardless of the quality of the health system and support attempts to introduce large-scale screening programs into the general population.

Poly(ADP-ribose) polymerase-1 dimerizes at a 5' recessed DNA end in vitro: a fluorescence study.

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation, or oxidants. The resulting formation of protein-bound poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA.