Structural transition in DNA and its effect on protein DNA interactions

Abstract

The demonstration of sequencedependent conformational flexibility of DNA and the discovery of lefthanded ZDNA paved the way for detailed studies on the possible role of such DNA structures. The monotonous double helix of Watson and Crick, which formed the foundation of modern molecular biology, has undergone dramatic conceptual changes through both theoretical and Xray crystallographic studies. To further our understanding of the factors controlling structural transitions in DNA, and the effects of DNA sequence/structurespecific control of events during gene expression, studies were carried out in our laboratory on certain model systems, forming the basis of this thesis. Spectroscopic and biochemical methods were employed to answer questions relating to structurespecific protein-DNA interactions. The questions addressed in this thesis concern:

the structural transition in DNA from B to Z form, its stabilization, and its effect on protein-DNA interactions.

The various aspects investigated include: a) Importance of critical cation balance in controlling the B Z transition in synthetic polynucleotides such as poly(dGdC); and the probable mode of stabilizing the Zform by low concentrations of multivalent coordination complexes. b) B Z transition and its effect on protein-DNA interactions, using the polymerase function of E. coli DNA polymerase I as a model system. c) Stabilization of altered structures (like ZDNA) in supercoiled plasmids and their preservation in the presence of Zstabilizing factors. d) Dynamic nature of the B Z transition under superhelical force. e) DNase I recognition and cleavage of ZDNA vs. BDNA. f) The probable role of DNA structural transitions in the origin of DNase I hypersensitivity and gene expression. Chapter 1 presents a brief overview of the major advances in our understanding of DNA structure. It includes a concise account of the present status of lefthanded ZDNA and the factors governing the B Z transition. A short account of DNA supercoiling, its effect on structural transitions, and its possible biological role is also provided. Monovalent ions such as Li, with high charge density and large hydration shells, are known to stabilize DNA in the Bform. The effect of Li in place of Na on the B Z transition of poly(dGdC) constitutes the first part of Chapter 2. Alcoholinduced B Z transition was absent in the Lisalt of poly(dGdC). In addition, the ethanolinduced transition in the Nasalt of poly(dGdC) was destabilized by Li. Mg² and hexammine cobalt chloride could compete out Li to induce the B Z transition in the Lisalt of poly(dGdC). The concentration of coordination complexes such as hexammine cobalt chloride required to stabilize ZDNA was found to be inversely proportional to the number of amine functions available for hydrogen bonding. Modelbuilding studies on ZDNA and hexammine cobalt chloride indicated the probable mode by which low concentrations of such complexes could stabilize the Zform. Chapter 3 investigates the effect of the B Z transition in synthetic polynucleotides such as poly(dGdC) and poly(dGdmC) on their ability to serve as templates for the DNA polymerase activity of E. coli DNA polymerase I. Poly(dGdC) in the Zform was found to be a poor template compared to the Bform. Additional studies under nearphysiological conditions with both polymers in B and Zforms showed a onetoone correlation between the B Z transition and decreased polymerase activity. The implications of these findings are discussed. Chapter 4 examines the role of supercoiling in modulating structural transitions and maintaining altered structures in circular DNA. To obtain DNA with different supercoiling levels, topoisomerase I was purified from wheat germ. To study structural stability mediated by transacting factors, a ZDNA binding protein was partially purified from wheat germ. Stabilization of ZDNA in circular plasmids by cobalt hexammine chloride was weaker than in linear synthetic polymers, but was enhanced by additional stabilization provided by ZDNA antibody. The ZDNA binding protein not only recognized Zsegments within natural sequences but also maintained these segments in the Zform even after superhelical tension was removed. This was studied by relaxing Zcontaining supercoiled plasmids with topoisomerase I in the presence of the binding protein. Such stabilization occurred only when the protein bound to the plasmid while the Zsegment was already in the Zform under supercoiling. Supercoilinduced B Z transition was found to be dynamic, as probed by restriction digestion of topoisomer populations using BssHII, which cannot cut ZDNA. The combined effects of cis and transacting factors on supercoiling and gene regulation are discussed. Hypersensitivity to DNase I has long been correlated with transcriptionally active genes and is believed to arise from altered DNA structures deviating from the Bform. Chapter 5 uses model systems to probe the structural prerequisites for DNase I action. ZDNA was completely resistant to DNase I digestion, in contrast to extensive cleavage in BDNA. A ZDNA stretch within a supercoiled plasmid also proved resistant, as shown by DNase I footprinting. Comparison of footprinting patterns between supercoiled and linearized plasmids revealed strong cleavage of BDNA but resistance of altered structures. The B-Z junction was found to be highly sensitive to DNase I. Even in the absence of protein factors, a transition from Z B conformation could produce DNase I hypersensitivity. Thus, hypersensitivity need not always require a structure other than BDNA; rather, structural polymorphism may suffice.