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UNWINDING THE DOUBLE HELIX: USING DIFFERENTIAL MECHANICS TO PROBE CONFORMATIONAL CHANGES IN 195 DNA measured in these experiments. This demonstrates that these analytical methods provide highly precise predictions of the details of strand separation in superhelical molecules. Figure 7.3 The transition profiles at DUE sequences. Reprinted, by permission, from Benham (1992). Copyright 1992 by Academic Press Limited. APPLYING THE METHOD TO STUDY INTERESTING GENES Having developed a method and confirmed its accuracy on test molecules, we can now apply it to study any DNA sequence of interest. It turns out to be particularly illuminating to examine the association between sites of superhelical destabilization and sites of gene regulation
UNWINDING THE DOUBLE HELIX: USING DIFFERENTIAL MECHANICS TO PROBE CONFORMATIONAL CHANGES IN 196 DNA (Benham, 1993). Our calculations show some striking correlations involving sites for initiation of transcription, termination of transcription, initiation of DNA replication, and binding of repressor proteins. We find that some bacterial genes show superhelical destabilization at the sites where gene expression starts and the sites where it ends. One gene on the pBR322 DNA molecule (from which the data in Figure 7.1 came) and one on the ColE1 plasmid (from which the data in Figure 7.4a came) are bracketed by such sites, suggesting that their expression is regulated by the state of DNA supercoiling. And, indeed, experiments show that these bracketed genes are expressed at higher rates when their DNA is superhelical than when it is relaxed. The other genes on these molecules show no such destabilized regions. This result suggests that genes in bacterial DNA can be partitioned into two categories, depending on whether or not they are bracketed by superhelically destabilized regulatory regions. In a similar vein, we have analyzed the DNA sequences of two mammalian viruses, the polyoma and papilloma viruses, each of which can cause cancer. The most destabilized locations on these molecules occur precisely at the places where gene expression terminates, the so-called poly-adenylation sites. The two most destabilized sites in the polyoma genome occur at the major (M) and minor (m) poly-adenylation sites, as shown in Figure 7.4b. Of the three most destabilized sites in the papilloma virus genome, two occur at known poly- adenylation sites for transcription from the direct strand. The other occurs at a location having the sequence attributes of a poly-adenylation site for transcription from the complementary strand. (This observation raises the intriguing possibility that the complementary strand of this molecule could transcribe, an event that has not been observed to date.) The strong association found between destabilized sites and the beginnings and ends of genes suggests that destabilization may play roles in their functioning. Many possible scenarios can be suggested for how this could occur. Clearly, destabilization at a gene promoter could facilitate the start of transcription by assisting the formation of a complex between the single strand to be transcribed and the enzyme complex that constructs the RNA transcript. What about destabilization at the sites where gene expression is completed (terminators in bacteria and poly-adenylation sites in higher organisms)? In this case, a likely but subtler role can be suggested. The moving transcription apparatus is thought to
UNWINDING THE DOUBLE HELIX: USING DIFFERENTIAL MECHANICS TO PROBE CONFORMATIONAL CHANGES IN 197 DNA Figure 7.4 The helix destabilization profiles of the circular molecules (top) ColEI plasmid DNA and (bottom) polyoma virus DNA. The locations of the promoter (P) and terminators (T1 and T2) of the bracketed transcription unit of ColEI are indicated. In polyoma the control region (denoted by a bar), replication origin (OR), and the major (M) and minor (m) poly-adenylation sites are shown. Reprinted, by permission, from Benham (1993). Copyright 1993 by the National Academy of Sciences. push a wave of positive supercoils ahead and leave a wake of negative supercoils behind (Wu et al., 1988). A region of strand separation constitutes a localized concentration of negative superhelicity, due to the large decrease in twist that occurs. This could provide a sink for the positive supercoils generated by an approaching complex, preventing the accumulation of twisting and bending deformations that otherwise could impede its progress. This would facilitate efficient transcription of the gene involved. The wake of negative supercoils left behind could destabilize the promoter region in preparation for the next round of expression. This model could explain why terminal regions are the most
UNWINDING THE DOUBLE HELIX: USING DIFFERENTIAL MECHANICS TO PROBE CONFORMATIONAL CHANGES IN 198 DNA destabilized sites found, and why some genes are bracketed by destabilized sites. DNA replication is another process for which it is interesting to study the correlation with superhelical destabilization. In the plasmids pBR322 and ColEI, replication is started by an RNA primer, which displaces one strand of the DNA at the replication origin by base pairing to the strand having a complementary sequence. The origin sites on these molecules are not destabilized by superhelicity, suggesting that the displacement event does not require a destabilized or separated site. By contrast, replication of the DNA of phage fl (a virus that attacks bacteria) involves enzymatic cutting of one strand that is known to require DNA superhelicity. If one role of DNA superhelicity is to promote strand separation, one would expect to find highly destabilized sites abutting the origin of replication. In fact, the calculations show precisely this, providing strong support for the assumption. Interesting results also emerge from the study of genes involved in the SOS response system in the bacterium E. coli. The SOS response system, as its name suggests, is a collection of genes that are turned on when the organism experiences any of a variety of serious problems, ranging from environmental stresses to DNA damage. These genes are usually turned off by the binding to their promoters of a repressor protein called LexA, which blocks transcription. Another protein, called RecA, plays a key role in initiating the SOS response by causing the removal of the LexA repressor, thereby allowing transcription. As it happens, the ColEI-encoded gene discussed above that was bracketed by superhelical destabilization sites is a member of the SOS response system. What about other SOS response genes? Do they also show superhelically destabilized regions? To address this question, we examined every known SOS response gene whose DNA sequence was available. In every case, the LexA binding site was contained in a strongly destabilized region. We note that this binding site is 16 base pairs long. Although it is reasonably A+Târich, this is not long enough for its presence alone to assure destabilization. It is not hard to speculate on the function of these superhelical destabilization regions in the SOS response. The RecA protein is known to bind single-stranded DNA. If the SOS response is marshaled against DNA damage, the damaged region provides single-stranded DNA for RecA binding. The environmental stresses that activate the SOS
