Poly(ADP-ribose) polymerase (PARP) is activated by binding to DNA strand breaks and contributes to various nuclear processes involving DNA strand breaks. This amended proposal aims to characterize the precise roles of PARP in DNA replication-repair and in apoptosis. It is based on the overall hypothesis that the covalent attachment of long, negatively charged chains of poly(ADP-ribose) (PAR) renders target DNA-binding proteins """"""""DNA-phobic"""""""" and that removal of PAR would then restore the affinity of these proteins for binding sites on DNA.
Specific Aim 1 A focuses on the role of PARP as a component of the multiprotein replication complex (MRC), or """"""""synthesome"""""""", which comprises about 40 proteins and is able to replicate, faithfully, SV40 DNA. MRCs derived from several cancer cell are error-prone. Several MRC components undergo poly(ADP-ribosyl)ation [p(ADP-R)n]; the use of cells from PARP knockout mice will be used to determine the function(s) of p(ADP-R)n on MRC proteins during DNA replication. To this end, the applicant will initially focus on one major facet--whether the MRC, purified from PARP knockout cells versus controls, exhibits a defect similar to cancer cells, that of error-prone replication.
Specific Aim 1 B focuses on E2F-1, which is normally induced during S phase of the cell cycle, but is not so expressed in cells from PARP knockout mice. The mechanism by which PARP regulates the promoter activity of this gene will therefore be investigated. In addition, the applicant will examine the direct interaction of PARP with the E2F-1 promoter directly, or in combination with other signal proteins that act with E2F-1, as well as direct or indirect effects of p(ADP-R)n on expression of E2F-1.
Specific Aim 2 is focused on the role of the early and transient p(ADP-R)n of nuclear proteins, described during the last grant period, which occurs early during apoptosis and is required for many of the subsequent changes characteristic of programmed cell death. The temporal and spatial relations of this transient p(ADP-R)n to changes in the structure of chromatin and the nuclear matrix will be examined systematically and compared, in part, by using PARP+/+ cells vs. PARP-/- cells to detect the function of p(ADP-R)n at this early critical point of apoptosis. The focus of Specific Aim 3 is the transcription factor and tumor suppressor p53, one of the major targets of p(ADP-R)n during apoptosis. The applicant identified p53 to be p(ADP-R)n briefly during the early burst of PAR in apoptosis. The subsequent cleavage of PAR from p53 coincides temporally with the activation of its target gene BAX. The mechanistic relation between this reversible modification of p53 and its transactivation activity and function will be determined in vitro and in the context of the whole cell by the use of gel shift assays and the activity of other p53 responsive genes.
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