Altered conformations in natural DNA: Studies on supercoiled form V DNA

Abstract

The double helical structure of DNA proposed by Watson and Crick, which led to the development of modern biology, was assumed to be a uniform right-handed helix. However, in recent years, the sequence-dependent conformational flexibility of DNA has been shown to be a rule rather than an exception through both theoretical and X-ray crystallographic studies on oligonucleotides. The discovery of left-handed Z-DNA paved the way for studies on the possible relevance of unusual secondary structures of DNA in biological processes.

Applying the topological properties, Weissmann and co-workers first predicted the possible existence of a left-handed structure within Form V DNA of natural sequences. So far, most of the structural studies on left-handed DNA have been done on synthetic oligonucleotides and polynucleotides of alternating purine-pyrimidine sequences, with little focus on naturally occurring sequences. Such a study requires natural DNA with a large amount of altered conformation, extending from right-handed B-DNA to left-handed Z-DNA under the influence of supercoiling.

The molecule of choice for the present study is Form V DNA with a linking number of zero. This is an in vitro reconstituted double-stranded circular DNA obtained by the reannealing of two single-stranded circles. Topological constraints require that every right-handed helical turn must be compensated by a left-handed helical turn or a negative supercoiling, since the linking number of Form V DNA is zero. This molecule adopts a negative superhelical density approaching 1.0. Therefore, several sequences are forced to adopt altered structures depending on the nature of the sequences themselves, as well as their neighboring sequences and torsional stress.

The questions raised in this thesis concern the altered conformations adopted by natural sequences in Form V DNA and their detection using sequence- and structure-specific probes. The various aspects investigated are:

i) Detection of the nature and extent of altered conformations in Form V DNA. ii) Interaction of altered conformations with structure-specific proteins, such as Z-DNA-specific antibody and Z-DNA-binding protein from wheat germ. iii) Effect of Z-DNA-stabilizing and DNA-unwinding agents on the stability of altered structures in Form V DNA. iv) Recognition of sequences present in altered conformation by sequence-specific restriction endonucleases. v) Accessibility of sequences in the altered conformations to methylation by modification methylases. vi) Capacity of sequences in natural DNA to adopt non-B conformations under topological strain. vii) Effect of flanking sequences on structural transitions. viii) Use of sequence-specific interactions to probe altered conformations in DNA and to rank such sequences based on their potential to adopt non-B conformation under the influence of topological unwinding.

Chapter 1 provides a brief account of major advances in our understanding of DNA structure. A concise account of the present status of left-handed DNA, various factors controlling B-to-Z structural transitions, and its probable biological role has been given. DNA supercoiling and its role in regulating genetic processes are also discussed. Studies carried out on Form V DNA by other workers are summarized in this chapter.

Chapter 2 describes the steps involved in the preparation and purification of Form V DNA from plasmids pFiG, and the monomer and dimer of pBR322. A brief account of various procedures used for the purification of supercoiled DNA, the starting material for Form V, is also provided.

Physicochemical and biochemical characterization of Form V DNA is presented in Chapter 3. Using two-dimensional electrophoresis and sedimentation methods, Form V DNA was found to possess high negative superhelical density. Spectroscopic studies indicated that more than 90% of the DNA is double-stranded, of which 35–40% is in left-handed conformation. A significant proportion of the left-handed segment is in the Z-DNA conformation, as indicated by binding of Z-DNA-specific protein and antibody. These left-handed structures in Form V DNA could be stabilized by hexamine cobalt chloride and destabilized by ethidium bromide.

To study the effect of altered conformation on sequence recognition by restriction endonucleases, cleavage of Form V DNA with various restriction enzymes was undertaken (Chapter 4). From studies with restriction endonucleases that have a single site in pBR322, it was found that recognition sequences for enzymes such as Aval, BamHI, and PstI were not accessible to cleavage in Form V DNA, indicating a probable alteration in structure. However, these sites became susceptible to cleavage when the topological constraints were relieved by simultaneous digestion either by topoisomerase or another restriction enzyme. This shows that altered DNA conformation is responsible for resistance to certain restriction endonucleases. Some sites showed partial cleavage. The possible nature of altered conformations adopted at various restriction sites is discussed.

In order to probe the altered conformations at regions within Form V DNA, two new methods were developed. Chapter 5 describes one method, which utilizes restriction endonucleases that have more than one site in pBR322. In this case, the nature of structures at various sites for FspI and Narl were assayed by partial cleavage of DNA followed by labeling at a specific site. In Form V as well as in Form I DNA, different sites showed various degrees of reactivity with the enzymes. However, Form V DNA exhibited greater resistance to cleavage compared to Form I DNA. The variable accessibility of sites is discussed in relation to structural alteration and the influence of flanking sequences in modulating such structural transitions. These studies provide strong evidence for the requirement of defined structures, in addition to sequence, for restriction by endonucleases, which can also explain the occurrence of slow sites in natural DNA.

A more efficient method for detecting structural alteration is presented in Chapter 6. This method utilizes sequence-specific DNA methylases as probes for B-DNA, exploiting the inability of methylases to methylate DNA adopting altered structures such as Z-DNA or single-stranded DNA. Since the DNA is not cleaved during methylation, the torsional stress of the molecule is maintained during treatment. Thus, the history of the structure at the methylatable site is preserved in terms of methylation level, even when the superhelical strain in Form V DNA is removed by subsequent restriction endonuclease cleavage. The nature and extent of altered conformations present at M.AluI, M.HhaI, and other sites are discussed. Some potential Z-forming alternating purine-pyrimidine sequences of fewer than seven base pairs were not in Z conformation, whereas others did adopt an altered structure, indicating an influence of neighboring sequences. Furthermore, regions of polypurine–polypyrimidine stretches were found to be the second-best candidates to adopt altered conformation.

Using the results from restriction endonuclease cleavage and methylation, a hierarchy of altered conformations adopted by DNA sequences in Form V was established, and a map indicating regions with structural alterations was developed. Thus, the combined influence of negative supercoiling and flanking sequences may have a strong effect on the structure adopted by sequences in vivo, thereby playing an important role in the regulation of genetic processes.

The work presented in this thesis, along with additional studies carried out by the candidate, has resulted in the following publications:

Left-handed DNA in synthetic and topologically constrained Form V DNA and its implications in protein recognition. Shouche, Y.S., Latha, P.K., Ramesh, N., Majumder, K., Mandyan, V., and Samir K. Brahmachari, J. Biosci. 8 (1985) 563–578.

Sequences that adopt non-B-DNA conformation in Form V DNA as probed by enzymatic methylation. Samir K. Brahmachari, Shouche, Y.S., Charles R. Cantor, and M. McClelland, J. Mol. Biol. 193 (1987) 201–211.

The influence of topological unwinding of DNA on restriction enzyme specificity. Shouche, Y.S., N. Ramesh, and Samir K. Brahmachari, (Submitted to Nucleic Acids Research)

Recognition of B- and Z-forms of DNA by E. coli DNA polymerase I. N. Ramesh, Yogesh S. Shouche, and Samir K. Brahmachari, J. Mol. Biol. 190 (1986) 635–638.

Stability of left-handed helical structure in pBR322 Form V DNA. Y.S. Shouche and Samir K. Brahmachari, (Manuscript in preparation)

Recognition of Z-DNA segments in Form V DNA by Z-DNA-binding protein from wheat germ. Y.S. Shouche, Eugenia T., N. Ramesh, and Samir K. Brahmachari, (Manuscript in preparation)