PARP REGULATED TRANSCRIPTION AND DNA DAMAGE RESPONSE

Georgetown University, Washington DC

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Abstract

DESCRIPTION (provided by applicant): Poly(ADP-ribose) polymerase (PARP) is critically involved in the immediate cellular response to DNA damage. Accumulating evidence has also implicated PARP in the regulation of transcription. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene regulating sequences in the absence of DNA damage. The overall objective of this proposal is to investigate the structure-functional relationships between PARP and promoter sequences in undamaged cells and in response to DNA damage induced by ionizing radiation. We have demonstrated that PARP can recognize and bind to DNA secondary structures, and to as yet unidentified sites in the PARP promoter sequences both in vitro and in vivo. Further, our previous studies provide support for PARP protein as a potent repressor of transcription and this function is attributable to the DNA-binding activity of PARP. Based on these observations, and the unique properties of PARP to automodify upon binding to broken DNA strands, we propose that PARP functions as a transcriptional silencer when recruited to promoters in living cells, and that DNA damage-induced auto(ADP-ribosyl)ation of PARP prevents its interaction with gene regulating sequences and subsequently alleviates a PARP-mediated block to transcription. We advance the hypothesis that PARP cycling between unmodified and poly(ADPribosyl) ated forms is an important component of the mechanism for regulating transcription in response to DNA damage. To test this hypothesis we will 1) define structural determinants for PARP binding to undamaged DNA using the model DNA constructs carrying stable secondary structures, 2) identify and map binding sites for PARP on its promoter in vitro, and 3) assay functional significance of PARP-promoter interactions in vivo in undamaged cells and in response to DNA damage using PARP gene transcriptional autoregulation as an experimental system. The completion of proposed studies will provide insight into molecular mechanisms activating cellular defense pathways in response to ionizing radiation and DNA damaging treatments. This mechanistic knowledge is important in support of other PARP-related studies (Projects 3 and 4 in this grant application) intended for future clinical translation.