Specific Binding of Poly(ADP-ribose) Polymerase-1 to Cruciform Hairpins Vladimir N. Potaman 1 * , Luda S. Shlyakhtenko 2 , Elena A. Oussatcheva 1 Yuri L. Lyubchenko 2 and Viatcheslav A. Soldatenkov 3 1 Institute of Biosciences and Technology, Texas A & M University System Health Science Center, 2121 W. Holcombe Blvd., Houston TX 77030, USA 2 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA 3 Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University Medical Center, 3970 Reservoir Rd, NW, Washington, DC 20007, USA Poly(ADP-ribose) polymerase-1 (PARP-1) participates in DNA cleavage and rejoining-dependent reactions, such as DNA replication, recombina- tion and repair. PARP-1 is also important in transcriptional regulation, although the determinants for its binding to undamaged genomic DNA have not been defined. Previously, we have shown by low-resolution mapping that PARP-1 may bind to the cruciform-forming regions of its own promoter. Here, using DNase I and nuclease P 1 footprinting and atomic force microscopy, we show that PARP-1 binds to stem/loop boundaries of 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. Furthermore, our data indicate that PARP-1 can interact with the gene regulatory sequences by binding to the promoter-localized cruciforms. q 2005 Elsevier Ltd. All rights reserved. Keywords: cruciform hairpin; DNA-binding; footprint; PARP-1; transcrip- tional regulation * Corresponding author Introduction Poly(ADP-ribose) polymerase-1 (PARP-1) (EC 2.4.2.30), an abundant nuclear protein, is involved in DNA functions where strand cleavage and rejoining takes place, such as DNA replication, recombination and repair. 1–3 A 46 kDa N-terminal DNA-binding domain (DBD) contains two putative zinc-fingers and two helix-turn-helix motifs and acts as a detector of DNA strand breaks. Being activated at DNA strand breaks, PARP-1 poly(ADP- ribosyl)ates itself and histones, thereby facilitating access to damaged DNA for reactions that restore the continuity of DNA strands. In addition to its function as a DNA damage sensor, PARP-1 may play important roles in transcriptional regulation by modulating changes in chromatin architecture, the poly(ADP-ribosyl)ation of transcription factors, and by direct binding to gene regulatory sequences. 4–7 PARP-1 is also involved in the autoregulation of gene expression by binding to certain elements in its own promoter. 8,9 Since PARP-1 has no apparent DNA binding sequence specificity, its regulatory function may be due to protein binding to non-B- DNA structures in the otherwise double-stranded B-DNA. In support of this suggestion, PARP-1 has been reported to bind to duplex crossovers, cruci- forms, bent DNA and base-unpairing regions, although little is known about the specific binding modes. 9–14 We have previously shown by chroma- tin immunoprecipitation assay and atomic force microscopy (AFM) that PARP-1 can bind to its promoter that contains inverted repeats (IRs) capable of cruciform formation. Incidentally, this region is also reactive to single-strand-specific nuclease P 1 , consistent with the presence of non-B- DNA structures. 9 Here, we used enzymatic and chemical probing as well as AFM to study PARP-1 binding to stable cruciform structures formed by model IRs and IRs from the PARP-1 promoter. Our results show that PARP-1 binds to the hairpin stem/loop boundaries but not the linear IRs. No appreciable PARP-1 binding to a four-way junction has been observed; however, PARP-1 binding at a distance appears to 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: PARP-1, poly(ADP-ribose) polymerase-1; IR, inverted repeat; AFM, atomic force microscopy; DMS, dimethylsulfate; Endo VII, T4 endonuclease VII; DBD, DNA-binding domain. E-mail address of the corresponding author: vpotaman@ibt.tamhsc.edu doi:10.1016/j.jmb.2005.03.010 J. Mol. Biol. (2005) 348, 609–615