Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n (CAG)n, (CGG)n (CCG)n, or (GAA)n (TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an intracellular sensor of DNA strand breaks and plays a critical role in cellular responses to DNA damage. In normally functioning cells, PARP-1 enzymatic activity has been linked to the alterations in chromatin structure associated with gene expression. However, the molecular determinants for PARP-1 recruitment to specific sites in chromatin in the absence of DNA strand breaks remain obscure. Using gel shift and enzymatic footprinting assays and atomic force microscopy, we show that PARP-1 recognizes distortions in the DNA helical backbone and that it binds to three-and four-way junctions as well as to stably unpaired regions in double-stranded DNA. PARP-1 interactions with non-B DNA structures are functional and lead to its catalytic activation. DNA hairpins, cruciforms, and stably unpaired regions are all effective co-activators of PARP-1 auto-modification and poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. Enzyme kinetic analyses revealed that the structural features of non-B form DNA co-factors are important for PARP-1 catalysis activated by undamaged DNA. K 0.5 constants for DNA co-factors, which are structurally different in the degree of base pairing and spatial DNA organization, 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 2-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 the normal physiology of the cell.Poly(ADP-ribose) polymerization is a post-translation protein modification that utilizes an ADP-ribosyl moiety from NAD ϩ to form branched polymers of up to 200 ADP-ribose units, which are attached via glutamic acid residues to the nuclear acceptor proteins. The best understood member of the superfamily of poly(ADP-ribose) polymerases (1) is PARP-1, 1 whose activity is largely accounted for by this type of nuclear protein modification (2). PARP-1 is an abundant zinc fingercontaining nuclear protein present at ϳ1 enzyme/50 nucleosomes. It has high affinity for damaged DNA and becomes catalytically active upon binding to double-and singlestranded DNA breaks (3). PARP-1 activation leads to modification of nuclear proteins including itself (auto-modification reaction) with a very strong polyanion, poly(ADP-ribose). This modification has a profound effect on the structure and function of the acceptor proteins. Based on these properties, PARP-1 has long been regarded as an intracellular sensor of DNA strand breaks, and its function has been considered in context with the cellular responses to genotoxic stress, in particular DNA damage repair and apoptosis (4 -7).In undamaged cells, recruitment of PARP-1 to the chromatin-modifying complex leads to a dramatic and localized perturbation of histone-DNA contacts (8, 9), allowing DNA to be accessible to regulatory factors, t...

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The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before a complete cleavage of four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving reaction conditions (Ca 2+ instead of Mg 2+ in the reaction buffer) was performed. Intramolecular complexes formed by the interaction with two binding sites in one DNA molecule (cis interaction) as well as the complexes obtained by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex the angles between the DNA strands at the complex proximity regardless of the complex type were systematically analyzed. All the data show a clear-cut bimodal distributions centered around peak values corresponding to 60° and 120°. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA templates with strands of different lengths and with different locations of the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles 60° or 120° between the strands corresponds to complex in which one of the strands flipped the orientation relative to another. Both types of complexes for 5 different sequences in the center are present almost equally. This finding suggests that there is no preferential orientation of the DNA cognate site within the complex suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein. KeywordsDNA synaptic complexes; protein-DNA interaction; restriction-modification; DNA looping; scanning probe microscopy Restriction enzyme SfiI belongs to a family of type II restriction endonucleases that bind and cut two cognate sites (type IIf, e.g., article (1) and references therein). A few enzymes of type IIf enzymes have been analyzed (Bse634I, Cfr10I, NgoMIV, NaeI and SfiI) and some details shedding a light on the mechanisms of the actions have been revealed. SfiI has been studied extensively by Halford and coworkers (recent paper (2) and references therein). † This research was supported by Grant GM 062235 (YLL) from the National Institute of Health. Crystallographic data have been obtained for Bse634I (3), NgoMIV (4) and Cfr10I (5). SfiI binds as a homotetramer to two recognition sites on DNA duplexes at the recognition sequence 5'-GGCCNNNN^NGGCC-3' (N denotes any base and ^ marks a cleavage position) prior to cleavage (6). The recognition regions can be in one DNA molecule or in two different molecules (cis and trans position, respectively) with higher reaction rate for cis position due to the higher effective local concentration of the interacting sites for the intramolecular reaction versus ...