Book of Abstracts: Albany 2005
PARP-1 Binding to Non-B-DNA Stimulates Auto- and Trans-poly(ADP-ribosyl)ation Reactions
Poly(ADP-ribose) polymerase-1 (PARP-1) participates in DNA cleavage and rejoining-dependent reactions, such as DNA replication, recombination, and repair. PARP-1 is also important in transcriptional regulation, although the determinants for its binding to undamaged genomic DNA have not been defined. We have previously shown by low-resolution mapping that PARP-1 may bind to the cruciform-forming regions of its own promoter. Here, we used enzymatic footprinting and atomic force microscopy to study PARP-1 binding to cruciforms and locally unpaired regions. Patterns of DNase I and P1 nuclease reactivity show that PARP-1 binds near the stem/loop boundaries of the cruciform hairpins. Cleavage of the cruciform by the junction resolvase, T4 endonuclease VII, is independent of PARP-1, which indicates that PARP-1 does not bind to the four-arm junctions of the cruciform. Thus, PARP-1 differs from other cruciform-binding proteins by binding to hairpin tips rather than to junctions. In the case of locally unpaired DNA, PARP-1 binds near the single/double strand DNA junction. The distances of PARP-1/DNA complexes from the ends of DNA fragments determined by AFM are consistent with protein binding to the cruciforms and unpaired regions. PARP-1 interactions with the non-B-DNA structures are functional: DNA hairpins, cruciforms, and stably unpaired regions are all effective activators of PARP-1 auto-modification and trans-poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. The enzyme kinetics analyses showed that PARP-1 catalysis activated by undamaged DNA appears to depend on the structural features of DNA co-factors: the values of K0.5 constants follow the order: cruciform ≤ hairpin << loop. DNA structure also influenced the reaction rate: when a hairpin was substituted with a stably unpaired region, the maximum reaction velocity decreased almost two-fold. These data suggest a link between PARP-1 binding to non-B-DNA structures in genome and its function in the dynamics of local modulation of chromatin structure in normal cell physiology.
Vladimir N. Potaman1,*
1Institute of Biosciences and Technology