DNA within living organisms occurs in a superhelical condition, which imposes stresses on the molecule. These stresses can destabilize the B-form duplex, causing local strand separations to occur at the sites where the thermodynamic stability is least. Theoretical methods have been developed to predict the locations and extents of destabilization in superhelical DNA sequences [1]. The results of these analyses agree precisely with experimental determinations of the extents and locations of denatured regions, as found by nuclease digestion [2]. This allows their use to predict the destabilization properties of other sequences, on which experiments have not been performed.

These methods have been applied to the analysis of genomic sequences from a wide range of organisms [3, 4]. The sites of predicted duplex destabilizations do not occur at random, but instead are closely associated with several specific types of DNA regulatory regions. The most strongly destabilized sites occur in the 3' flanks of genes. This pattern is detected in prokaryotes, eukaryotic viruses, yeast and humans. An analysis of 26 yeast genes found that their promoter and terminal regions were destabilized, but the region encoding the primary transcript was not. This same tripartite pattern is found in rDNA sequences, which are transcribed by a different polymerase.

Origins of replication contain regions that are susceptible to stress-induced strand separation. The autonomously replicating sequences (ARSs) in the yeast genome require a destabilized site at a specific location to be active.

The third class of regulatory regions that exhibit characteristic destabilization properties are sites of DNA attachment. Scaffold attachment regions (SARs), which are sites where the chromatin fiber is attached to the chromosomal matrix, contain sites of stress-induced destabilization. The centromere regions of two yeast species (baker's yeast and fission yeast) both contain sites where the strands of the DNA duplex separate under imposed stress. It appears that the single strands within this region form hairpin structures that are involved in kinetochore binding [5].

This talk will briefly describe the techniques used to analyze duplex destabilization in superhelical DNA. A selection of predictions regarding the destabilization properties of these three classes of regulatory regions will be presented. The implications of these results concerning possible regulatory mechanisms will be considered.

The strong associations found between stress-destabilized sites and specific categories of regulatory regions suggests that the presence of such sites may be necessary for function. Experimental results support this conclusion in several specific cases. This suggests that methods to evaluate the destabilization properties of putative regulatory regions may be useful in discriminating which sites are active. The incorporation of these methods into strategies to search genomic sequences for regulatory regions will be discussed.

References:

1. C.J. Benham (1990) Theoretical Analysis of Heteropolymeric Transitions in superhelical DNA Molecules of Specified Sequence, J. Chem. Phys. 92: 6294-6305.
2. C.J. Benham (1992) Energetics of the Strand Separation Transition in Superhelical DNA, J. Mol. Biol. 225: 835-847.
3. C.J. Benham (1993) Sites of Predicted Stress-Induced DNA Duplex Destabilization Occur Preferentially at Regulatory Loci, Proc. Nat'l. Acad. Sci. USA 90: 2999-3003.
4. C.J. Benham (1996) Duplex Destabilization in Superhelical DNA is Predicted to Occur at Specific Transcriptional Regulatory Regions, J. Mol. Biol. 255: 425-434.
5. M. Tal, F. Shimron and G. Yagil (1994) Unwound Regions in Yeast Centromere IV DNA, J. Mol. Biol. 243: 179-189.

Back to Workshop Schedule

1996-1997 Mathematics in High Performance Computing