| dc.description.abstract | Since the discovery of DNA and the proposal of the double helix structure, we have come a long way in understanding the molecular basis of heredity. With the ability to synthesize any DNA sequence of interest and improved methods for the study of DNA structure, we now know that in addition to the conformational flexibility observed within the B-DNA definition, DNA can also form several different structures, such as left-handed DNA, triple helix, and quadruplex. It has been shown in recent years that in addition to the sequence information for coding amino acids, the sequence of DNA contains higher-order physical information pertaining to shape and topology. Several sequences have been shown to undergo transition to non-B structures. Factors that facilitate structural transitions in a given DNA sequence, such as ionic conditions, supercoiling, and protein interactions, are also well documented in the literature.
With the discovery of such non-B potential sequences in the DNase I hypersensitive regions of active genes, these structures assumed new importance. Subsequently, attempts have been made to demonstrate at least some of these structures in vivo. All this was valid for the prokaryotic system. When we consider eukaryotes, we encounter two additional features. Firstly, the eukaryotic genome exists as chromatin, and secondly, it is rich in repetitive sequences. The nucleosomal DNA is wound around the basic histone core in 1.75 turns of a DNA superhelix. Since the bendability of a DNA sequence is inbuilt in its base sequence, the sequence of a stretch of DNA determines the ease with which it can accommodate a nucleosome. This led to detailed studies on nucleosome phasing.
In addition, several DNA-binding proteins have been discovered that bind to particular sequences or conformations of DNA. Thus, besides the codon sequence that dictates the amino acid sequence of the protein it codes for, a given DNA sequence has structural information. This structural information determines not only the conformation adopted by the DNA sequence itself but also its ability to form a nucleosome and interact with other proteins.
An examination of the non-coding DNA of eukaryotes revealed that several of these sequences are repetitive in nature. Many simple repetitive sequences satisfy the sequence criteria for forming a non-B structure. Our laboratory has been interested in studying the ability of natural DNA sequences to adopt unusual DNA structures. The ability of some of these structures to affect gene expression was being investigated. We were interested in studying the ability of these repetitive sequences to organize chromatin. Though several reconstitution studies had been carried out on synthetic polynucleotides, little was known about the in vivo status of these repetitive sequences in the genome.
With our current knowledge of the existence of topological domains in eukaryotic chromatin, the presence and possible role of supercoil-stabilized unusual structures in chromatin organization assumed special significance. Towards this end, we carried out the present study.
These are the questions we have addressed in this thesis:
Can naturally occurring d(TG)n repeats present within introns adopt altered conformations?
Are repetitive DNA sequences like the Z-potential d(TG)n sequences, the triple helix potential d(TC)n, and the quadruplex potential telomeric sequences nucleosomal in vivo?
Do such repetitive sequences, by virtue of either sequence or structure, differ from bulk DNA in their association with the histone octamer?
Will a highly supercoiled molecule like form V DNA, which has unusual structures distributed all over the molecule, allow nucleosome formation?
A brief description of our investigations towards answering the above questions follows.
In Chapter 1, we have reviewed the current knowledge of repetitive DNA, supercoiling, and DNA structures. Our present understanding of chromatin structure and nucleosome positioning has also been described. The chapter details our perspective of the eukaryotic genome and our objective in carrying out the present study.
In Chapter 2, the widespread occurrence of a simple repeat, the d(TG)n sequence, with respect to various genes is discussed. We have studied the 0.9 kb sequence of the third intron of the rat ?-lactalbumin gene, which has two Z-potential stretches d(TG)14 and d(TG)24 separated by 318 bp. By two-dimensional chloroquine gel electrophoresis, we show that these two sequences adopt Z-structure at different superhelical densities. The presence of Z-structure was confirmed by gel mobility retardation studies with Z-22, a monoclonal anti Z-DNA antibody. In addition, we propose that the B to Z transition is affected not only by the length of the potential Z-stretch but also by the size of the domain in which it exists and the twist-absorbing capacity of the domain.
In Chapter 3, we have detailed our study of chromatin organization of two repetitive sequences, the Z-potential d(TG)n sequence and the triple helix potential d(TC)n sequence, in rat liver nuclei. By hybridization of Southern blots of micrococcal nuclease digests, we show that these two sequences differ in their ability to organize nucleosomes.
We have adopted the in vivo chemical crosslinking and protein image hybridization technique of Mirzabekov et al. on rat liver nuclei. In Chapter 4, we describe how we have exploited this technique to study DNA-histone association in vivo. Methylation of purines is followed by depurination, resulting in the generation of an active aldehyde group. A Schiff’s reaction between proximal histone functional groups and such active aldehyde groups causes the generation of a covalent bond accompanied by breaks in the sugar-phosphate backbone. Thus, a DNA strand gets cross-linked to proximal protein imidazole and amino functional groups. After several purification steps to enrich the cross-linked DNA, it can then be separated from free DNA by two-dimensional denaturing gel electrophoresis. This resolved genomic DNA can then be hybridized using probes of sequences of interest.
We performed such protein image hybridizations for repetitive DNA sequences to look at differences in histone-DNA contacts for these simple repeats. We find that both d(TG)n and d(TC)n sequences have cross-linkable histone domains in proximity to histone functional groups and are not nucleosome-free. However, there appeared to be differences in the distribution of intensities in the crosslinked DNA diagonal for each of these repeats. The kind of DNA-histone association might determine the distribution of signal intensity in these diagonals.
The telomeric sequences, which occur at the physical ends of chromosomes, are able to form quadruplex structures in vitro. Our study on the chromatin organization of the vertebrate telomeric sequence in rat liver nuclei, using both micrococcal nuclease digestion and chemical crosslinking followed by protein image hybridization, is detailed in Chapter 5. Our micrococcal nuclease digestion data revealed that these sequences are apparently not easily accessible for digestion at early time points. However, as digestion proceeds, these sequences come into the soluble fraction and exhibit protection of a classical core particle. The protein image hybridization data for the sequence suggested a different organization for each strand of the telomeric sequence. Though the C-rich strand showed hybridization to the crosslinked diagonal, the G-rich strand was apparently devoid of any histone contacts. Since the chemical modification by dimethyl sulfate requires a free N7 of guanine for methylation, the lack of crosslinking might be due to the formation of unusual structures even in vivo. The presence of Hoogsteen base pairs in vitro prevents methylation of the G-residues in the telomeric oligonucleotide, which adopts a folded quadruplex structure. It, therefore, suggested that these G-rich telomere sequences might form similar unusual structures even in vivo.
All of the above studies were carried out in vivo, where we were able to study the chromatin organization of repeat sequences of interest. But how do nucleosomes interact with alternate structures? Do they favor or inhibit their formation? In order to directly correlate DNA structure with chromatin organization, we undertook in vitro reconstitution studies on the form V DNA molecule. This molecule is made by reannealing two complementary single-stranded circles. Since it has zero linking number, the molecule has a high amount of supercoiling, with many unusual structures distributed throughout the molecule.
Using the pBR322 form V molecule as a model system, the relationship between DNA sequence and structure has been extensively studied in our laboratory earlier. The structure sensitivity of restriction enzymes has been exploited to map the altered structures all over this molecule. In Chapter 6, we present our nucleosome reconstitution data on this form V molecule. Surprisingly, despite the presence of a large extent of altered structures distributed throughout the molecule, we found that form V DNA can accommodate nucleosomes. Restriction analysis showed that some of the restriction sites which were inaccessible in the pBR322 form V DNA molecule remained inaccessible even after reconstitution with core histones. This implied that the supercoils absorbed by the reconstituted nucleosomes were not sufficient to cause reversal to the accessible B-form at these sites. Addition of form V DNA to reconstituted form I molecule resulted in the transfer of some of the core histones to the more negatively supercoiled form V molecule.
Finally, we propose a hypothesis for the generation of supercoils in the absence of any gyrase-like molecule in the eukaryotic system. We postulated that variation in nucleosomal density and the ability of nucleosomes to move between segments of repetitive DNA and random DNA can regulate supercoiling levels in eukaryotes. Based on our data of the chromatin organization of repetitive DNA, we propose that the unique ability of each of these sequences to organize chromatin might serve as signals for regulatory proteins in the nucleoprotein milieu of the nucleus. | |
