XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage.

The two BRCT domains (BRCT1 and BRCT2) of XRCC1 mediate a network of protein-protein interactions with several key factors of the DNA single-strand breaks (SSBs) and base damage repair pathways. BRCT1 is required for the immediate poly(ADP-ribose)-dependent recruitment of XRCC1 to DNA breaks and is essential for survival after DNA damage. To better understand the biological role of XRCC1 in the processing of DNA ends, a search for the BRCT1 domain-associated proteins was performed by mass spectrometry of GST-BRCT1 pulled-down proteins from HeLa cell extracts. Here, we report that the double-strand break (DSB) repair heterotrimeric complex DNA-PK interacts with the BRCT1 domain of XRCC1 and phosphorylates this domain at serine 371 after ionizing irradiation. This caused XRCC1 dimer dissociation. The XRCC1 R399Q variant allele did not affect this phosphorylation. We also show that XRCC1 strongly stimulates the phosphorylation of p53-Ser15 by DNA-PK. The pseudo phosphorylated S371D mutant was a much weaker stimulator of DNA-PK activity whereas the non-phosphorylable mutant S371L endowed with a DNA-PK stimulating capacity failed to fully rescue the DSB repair defect of XRCC1-deficient EM9 rodent cells. The functional association between XRCC1 and DNA-PK in response to IR provides the first evidence for their involvement in a common DSB repair pathway.

The PARP superfamily.

Poly(ADP-ribosyl)ation is an immediate DNA-damage-dependent post-translational modification of histones and other nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a large family of 18 proteins, encoded by different genes and displaying a conserved catalytic domain in which PARP-1 (113 kDa), the founding member, and PARP-2 (62 kDa) are so far the sole enzymes whose catalytic activity has been shown to be immediately stimulated by DNA strand breaks. A large repertoire of sequences encoding novel PARPs now extends considerably the field of poly(ADP-ribosyl)ation reactions to various aspects of the cell biology including cell proliferation and cell death. Some of these new members interact with each other, share common partners and common subcellular localizations suggesting possible fine tuning in the regulation of this post-translational modification of proteins. This review summarizes our present knowledge of this emerging superfamily, which might ultimately improve pharmacological strategies to enhance both antitumor efficacy and the treatment of a number of inflammatory and neurodegenerative disorders. A provisional nomenclature is proposed.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells.

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms

Entry inhibitors SCH-C, RANTES, and T-20 block HIV type 1 replication in multiple cell types.

The small-molecule CCR5 antagonist SCH-C (SCH 351125) was tested for its ability to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells, immature dendritic cells (DCs), and macrophages. Inhibition of infection of PBMCs by virus associated with mature DC in trans was also studied. For comparison, the peptide-based fusion inhibitor T-20 and the CC-chemokine RANTES were also evaluated. Although some cell type-dependent differences in potency were observed, each of the three entry inhibitors was active against the replication of three different CCR5-using primary isolates in each cell type. CCR5-dependent HIV-1 infectivity, whether DC associated or not, is thus vulnerable to inhibitors that block the virus-cell fusion process by different mechanisms. Together, these results suggest that SCH-C and other entry inhibitors should be evaluated for their clinical potential as inhibitors of HIV-1 replication in several settings, including the prevention of maternal-infant transmission and the prevention of sexual transmission by topical application as a microbicide.

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression.

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

Human immunodeficiency virus type 1 attachment, coreceptor, and fusion inhibitors are active against both direct and trans infection of primary cells.

Inhibitors of human immunodeficiency virus type 1 attachment (CD4-immunoglobulin G subclass 2), CCR5 usage (PRO 140), and fusion (T-20) were tested on diverse primary cell types that represent the major targets both for infection in vivo and for the inhibition of trans infection of target cells by virus bound to dendritic cells. Although minor cell-type-dependent differences in potency were observed, each inhibitor was active on each cell type and trans infection was similarly vulnerable to inhibition at each stage of the fusion cascade.

Nonneutralizing antibodies to the CD4-binding site on the gp120 subunit of human immunodeficiency virus type 1 do not interfere with the activity of a neutralizing antibody against the same site.

We have investigated whether nonneutralizing monoclonal antibodies (MAbs) to the gp120 subunit of the envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 (HIV-1) can interfere with HIV-1 neutralization by another anti-gp120 MAb. We used neutralizing (b12) and nonneutralizing (205-42-15, 204-43-1, 205-46-9) MAbs to the epitope cluster overlapping the CD4-binding site (CD4BS) on gp120. All the MAbs, neutralizing or otherwise, cross-competed for binding to monomeric gp120, indicating the close topological proximity of their epitopes. However, the nonneutralizing CD4BS MAbs did not interfere with the neutralization activity of MAb b12. In contrast, in a binding assay using oligomeric Env expressed on the surface of Env-transfected cells, the nonneutralizing MAbs did partially compete with b12 for Env binding. The surface of Env-transfected cells contains two categories of binding site for CD4BS MAbs. One type of site is recognized by both b12 and nonneutralizing CD4BS MAbs; the other is recognized by only b12. Binding assays for Env-gp120 interactions based on the use of monomeric gp120 or Env-transfected cells do not predict the outcome of HIV-1 neutralization assays, and they should therefore be used only with caution when gauging the properties of anti-Env MAbs.