We determined the magnitude of the bend induced in DNA by an adenine-thymine tract by measuring the rate of cyclization of DNA oligonucleotides containing phased A tracts. A series of linear multimers with 2-bp single-stranded ends, in which the (A.T)6 tracts are separated by CG2-3C sequences and are positioned 10 and 11 bp apart alternately, were prepared from 21 bp long synthetic duplexed deoxyoligonucleotides. The cyclization rates of the multimers (105-210 bp) and the bimolecular association rate of the 84 bp long multimer were measured in the presence of DNA ligase. From the rate constants of the cyclization and bimolecular association reactions, ring closure probabilities were obtained for the multimers. The systematically bent molecules were simulated by Monte Carlo methods, and the ring closure probabilities were calculated for a given set of junction bend angles. By comparing the calculated values of ring closure probabilities to experimental values and adjusting the junction bend angles to fit experimental values, the extent of bending at the junctions (or the extent of bending for an adenine tract) was determined. We conclude that an A6 tract bends the DNA helix by 17-21 degrees.

We have characterized structural and dynamic aspects of the d(C1G2CsG4AS&T6TSC4G3C2G,) 12-mer du-

No abstract

These results provide a first look into the structure and catalytic residues of a non-metal dehydratase, a large class of hitherto structurally uncharacterized enzymes. It is envisaged that a detailed structural description of scytalone dehydratase will assist in the design of new inhibitors for controlling rice blast disease.

We used gel electrophoresis methods to show that reaction of DNA with cis-diamminedichloroplatinum (II) results in a substantial (--40°) bend in the double helix at the intrastrand crosslink between the N7 atoms of adjacent guanosine nucleosides, which bond to the platinum(II) complex with loss of two chloride ions. Multimers of a 22-base-pair (bp) oligonucleotide platinated at a single site show strong anomalies in their electrophoretic mobilities as a consequence of coherent addition of in-phase platinum-induced bends. Increase of the sequence repeat of the platinated site to 27 bp yields multimers of nearly normal mobility because the bends are out of phase. MATERIALS AND METHODS Oligonucleotides were synthesized on an Applied Biosystems model 380B DNA synthesizer and purified by reversephase HPLC. The strand (Fig. 1) containing adjacent guanines was platinated with cis-[Pt(NH3)2(H2O)2 ' by standard methods (16). After the crude reaction mixture was purified by reverse-phase HPLC, the single major product was shown by flameless atomic absorption spectroscopy to have 0.98 platinum atom per strand. From the sequence, which was chosen to contain two adjacent guanosines and no other purines, and from the known chemistry of cisplatin with oligonucleotides (5), this adduct may be safely concluded to be the intrastrand d(pGpG) crosslink. Complementary strands were labeled with 32P at the 5' terminus, hybridized, and ligated as described (14).RESULTS AND DISCUSSION Multimers of the platinated 22-base-pair (bp) duplex oligonucleotide shown in Fig. 1, designated P, were used to investigate Pt-induced DNA bending. Since cisplatin unwinds the DNA double helix (17)(18)(19), an 11-bp repeat (slightly more than the normal helix repeat of 10.5 bp) was selected to optimize the match between the sequence repeat and the expected helix screw. The ligation reaction yielded a mixture of circular and linear oligomers; these products separated into two intersecting ladders in nondenaturing polyacrylamide electrophoresis gels (Fig. 2). The smallest circular product (CS) was excised from the gel, denatured, and run on a 20%6 (20:1, acrylamide/methylenebisacrylamide) denaturing gel containing 7 M urea. The sample contained predominantly intact and oncenicked single strands (data not shown); the mobility of the latter corresponded to a length of 110 bp, representing a 5-mer of the original monomer sequence. The predominance of circular products for multimers larger than 100 bp is consistent with strong DNA bending and confirms that there is a close match between the 22-bp sequence repeat and the helix screw (20, 21).The comparative mobilities of different-length fragments generated by self-ligation of the 22-bp platinated monomer P are plotted in Fig. 3. The ratio RL of apparent size (determined by comparison with the BamHI ladder) to true size increases dramatically with DNA length, resembling the electrophoretic behavior ofbent DNA molecules in which the sequence repeat closely matches the double helix screw repeat (14). ...