No abstract

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase ␦ assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21 waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.1 is a DNA-nick sensor protein that uses ␤-NAD ϩ as a substrate for transferring ADP-ribose moieties to itself and to nuclear acceptor proteins (1). PARP-1 modulates the structure and function of many proteins involved in DNA metabolism (2, 3), co-purifies with some members of the DNA synthesome (4 -6), and is a component of replication-competent complexes (7). It has been shown previously that PARP-1 co-immunoprecipitates with the proliferating cell nuclear antigen (PCNA) (4, 5), which is a pivotal protein in DNA replication, DNA repair, and cell cycle control. A number of proteins involved in DNA replication and repair interact with PCNA (reviewed in Refs. 8 -11). Most of the PCNA-interacting proteins have a QXX(h)XX(a)(a) box that specifically binds the interdomain connector loop (12). PARP-1 shows a putative PCNA-binding consensus sequence (QDLIK-MIF) at position 669 within the NAD-binding domain that is essential for the conversion of NAD into ADP-ribose and, consequently, for PARP-1 catalytic activity.In this work we sought to attain a greater understanding of the interaction between PARP-1 and PCNA. We have demonstrated that PARP-1 and PCNA co-immunoprecipitate both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. These results were supported by immunoprecipitation experiments with purified proteins. To investigate the effect of this association on the properties of each protein, we evaluated the conversion of NAD into ADP-ribose (PARP assay) as well as PCNA-dependent nucleotide incorporation (pol ␦ assay), and we found a marked inhibition of both reactions. Unexpectedly, an inhibitory effect on PARP-1 activity was al...

No abstract

The ability of the cyclin-dependent kinase (CDK) inhibitor p21 CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G 1 phase cell cycle arrest induced by p21GFP occurred also at the G 1 /S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21 PCNA-) or CDK (p21 CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21 wt HA and p21 CDK -HA, but not p21 PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase δ (pol δ). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol δ to PCNA at the G 1 /S phase transition.

Free-ranging sea otters are subject to hydrocarbon exposure from a variety of sources, both natural and anthropogenic. Effects of direct exposure to unrefined crude oil, such as that associated with the Exxon Valdez oil spill, are readily apparent. However, the impact of subtle but pathophysiologically relevant concentrations of crude oil on sea otters is difficult to assess. The present study was directed at developing a model for assessing the impact of low concentrations of fuel oil on sea otters. Quantitative PCR was used to identify differential gene expression in American mink that were exposed to low concentrations of bunker C fuel oil. A total of 23 genes, representing 10 different physiological systems, were analyzed for perturbation. Six genes with immunological relevance were differentially expressed in oil-fed mink. Interleukin-18 (IL-18), IL-10, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and complement cytolysis inhibitor (CLI) were down-regulated while IL-2 was up-regulated. Expression of two additional genes was affected; heat shock protein 70 (HSP70) was up-regulated and thyroid hormone receptor (THR) was down-regulated. While the significance of each perturbation is not immediately evident, we identified differential expression of genes that would be consistent with the presence of immune system-modifying and endocrine-disrupting compounds in fuel oil. Application of this approach to identify effects of petroleum contamination on sea otters should be possible following expansion of this mink model to identify a greater number of affected genes in peripheral blood leukocytes.