Heterogeneity in the actions of drugs that bind in the DNA minor groove.

Distamycin and Hoechst 33258 have long served as the model compounds for biochemical, biophysical, and clinical studies of the drugs that bind in the DNA minor groove. However, the results presented in this investigation clearly show that 4,6-diamidino-2 phenylindole (DAPI) is superior to both of these drugs at negating the effects of intrinsic DNA curvature and anisotropic bendability as measured by electrophoretic and ligation analysis. In addition, DAPI was more effective than distamycin and Hoechst 33258 at inhibiting the assembly of nucleosomes onto synthetic and natural sequences that have multiple closely spaced oligo-AT sequences that serve as drug binding sites. Since these effects may be related to the biological action of the drugs, it was of interest to determine the mechanism that was responsible for the enhanced action of DAPI. The possibility that the differential drug potencies resulted from differential overall affinities of the ligands for A-tract molecules was considered, but drug binding studies suggested that this was not the case. It is also unlikely that the differential drug effects resulted from the binding of the drugs to different DNA sites since the oligo A/T binding sites for DAPI and Hoechst were centered on the same nucleotide positions as revealed by footprinting studies using exonuclease III, DNase I, and hydroxyl radical. However, the footprinting studies with DNase I did uncover a potentially important difference between the drugs. DAPI protected only the AT bp in the binding sites, while distamycin and Hoechst protected these bp as well as flanking Gs and Cs. These results permitted us to advance a preliminary model for the enhanced action DAPI. According to the model, the short length of DAPI and its absolute specificity for A/T bps with narrow minor grooves ensures that only particularly minor grooves that give rise to curvature and anisotropic bendability are occupied by the drug. Consequently, each helical deflection induced by an A-tract in the absence of the drug is countered by an opposite deflection induced by DAPI binding, thus effectively neutralizing intrinsic curvature and bending into the minor groove.

Conserved DNA structures in origins of replication.

According to the model of Bramhill and Kornberg, initiation of DNA replication in prokaryotes involves binding of an initiator protein to origin DNA and subsequent duplex opening of adjacent direct repeat sequences. In this report, we have used computer analysis to examine the higher-order DNA structure of a variety of origins of replication from plasmids, phages, and bacteria in order to determine whether these sequences are localized in domains of altered structure. The results demonstrate that the primary sites of initiator protein binding lie in discrete domains of DNA bending, while the direct repeats lie within well-defined boundaries of an unusual anti-bent domain. The anti-bent structures arise from a periodicity of A3 and T3 tracts which avoids the 10-11 bp bending periodicity. Since DNA fragments which serve as replicators in yeast also contain these two conserved structural elements, the results provide new insight into the universal role of conserved DNA structures in DNA replication.

DNA structures associated with autonomously replicating sequences from plants.

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.

Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.

Computer modelling of DNA structures involved in chromosome maintenance.

Sequence-dependent DNA bending of synthetic and natural molecules was studied by computer analysis. Modelling of synthetic oligonucleotides and of 107 kb of natural sequences gave results which closely resembled published electrophoretic data, demonstrating the powerful predictive capacity of the procedure. The analysis was extended to the study of DNA structures involved in chromosome maintenance. Centromeric DNAs from yeast were found to have sequences in their functional elements which cause them to be unusually straight. Autonomous replicating sequences were found to have two structural domains, one consisting of unusually straight sequences surrounding the consensus and the other of bending elements in flanking DNA. In addition to a structural homology, centromeric and autonomous replicating sequences share common sequence elements. These observations show that computer modelling of natural sequences is a viable approach to the study of the biological implications of alternative DNA structures.