PARP I is the acronym for an abundant nuclear enzyme that catalyzes the formation of poly ADP-ribose chains on itself and other nuclear proteins. PARP I is known to be a multi-functional enzyme but only one of its roles has been analyzed in detail, namely its super-activation in response to DNA damage, the consequence of which is to contribute to apoptosis or cell death. This pathophysiolgical role has overshadowed its other physiological roles, which have been suggested to involve a variety of cellular activities such as DNA replication, gene transcription, and chromatin assembly. It also links these activities with the dynamics of cellular oxidative metabolism via its substrate, NAD. To date, no coherent model that accounts for PARP I's involvement in these cellular activities has emerged. One central barrier to the understanding of PARP I's normal (ie non-pathological or apoptotic) role has been the widely held belief that it requires """"""""broken"""""""" or """"""""damaged"""""""" DNA (DNA with single- or double-stranded breaks) as an obligatory coenzyme. DNA in normal chromatin is unbroken. So, how could PARP I be activated in the nucleus of a normal cell unless DNA breakage occurs? The work performed under this grant application has discovered, for the first time, that the intact linear double-stranded DNA (dsDNA) serves as an efficient coenzyme for PARP I activity. In fact, we have shown that the affinity of PARP I for coenzymic dsDNA is 100-fold stronger than for """"""""damaged"""""""" (DNase-treated) DNA. Therefore, PARP I's function in normal cells does not require single strand nicks in chromosomal DNA. To the contrary, PARP I prefers the normal chromatin form of DNA (ie. dsDNA) as a coenzyme, and this form of DNA can unequivocally be stated to be the major coenzyme for PARP I activity in normal cells. Histone H1 is a powerful activator of PARP-1 and is also a major acceptor (trans-ADPRn modification) as is PARP-1 itself (auto-ADPRn modification). Both activities of histone H1 are likely to have profound effects on the chromatin structure and remodeling, but they are not yet defined. Two key observations indicate that skeletal muscle differentiation is a system, in which PARP-I's physiological function(s) can be elucidated. First, we have shown that PARP-1 is the key regulatory protein responsible for activation of one contractile protein gene in skeletal muscle cells and for the repression of this gene in all non-muscle cell types. PARP- 1 accomplishes this through binding to a specific nucleotide sequence motif as well as through its enzymatic activity. We hypothesize that PARP-1 may be stably bound to chromosomal DNA at many such sites and exert its gene regulatory role via localized enzymatic activity. Second, inhibition of PARP-1 activity blocks myogenic differentiation at a stage preceding contractile protein expression. This block is reversible upon removal of the inhibitors. Thus, PARP-1 activity is necessary for progression of myoblasts to myocytes. The mechanistic bases for both findings are incompletely understood. The proposed research will focus on the biochemical and molecular basis for the PARP-I's role in muscle differentiation and muscle gene regulation, and the results will, for the first time, define a physiological role of this abundant yet mysterious chromosomal protein.
