Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 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,*
Irina A. Lonskaya2
Luda S. Shlyakhtenko3
Elena A. Oussatcheva1
Yuri L. Lyubchenko3
Viatcheslav A. Soldatenkov2

1Institute of Biosciences and Technology
Texas A&M University System Health Science Center
2121 W. Holcombe Blvd.
Houston, TX 77030
2Department of Radiation Medicine
Lombardi Cancer Center
Georgetown University Medical Center
3970 Reservoir Rd.
NW, Washington, DC 20007
3Department of Pharmaceutical Sciences
University of Nebraska Medical Center
Omaha, NE 68198