Graduate Thesis Or Dissertation
 

Linker histone/DNA interactions : in vitro studies

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  • By studying the linker histone/DNA complex in vitro, our goal was to gain further insight into the interaction of these histones in chromatin structure, which, in turn, helps us in better understanding critical biological processes such as replication, transcription, and recombination. In direct competition experiments, we have confirmed previous results of the preference of the linker histones in binding to superhelical DNA over linear or nicked circular DNA forms. This binding of linker histones to DNA supercoils in the presence of DNA competitors was examined at varying histone/DNA ratios and different ionic strengths. It was demonstrated that: 1) With increasing H1/DNA ratios, the electrophoretic mobility of the H1/supercoiled DNA complex decreases. 2) With increasing ionic strengths, the mobility of this complex increases. The presence of more protein bound to superhelical DNA explains the first observation. The second observation, however, is explained by a reduction in the amount of protein bound to DNA as well as a change in the conformation of the complex at higher salt concentrations. The conformational change of the superhelical DNA upon linker histone binding was then investigated. The effect of linker histone binding on the accessibility of sites on superhelical plasmids to single-strand-specific nucleases (P1 or S1) was studied first. The results show protection of preexisting P1- or S1- sensitive sites at low to moderate linker histone/DNA ratios as well as the appearance of new susceptible sites at higher ratios. The protection of singlestrand-specific nuclease-sensitive sites can be explained by a change in the superhelical torsion of the plasmid as a result of linker histone binding. Furthermore, the interaction of the C-terminal domain of the linker histones with AT-rich sites on DNA could (by destabilizing B-DNA structure) make the sites susceptible to nuclease cleavage. This explains the appearance of new susceptible sites at higher linker histone levels. These changes in the pattern of cleavage by nucleases with increasing linker histone/DNA ratios were further studied by investigating the effect of linker histones on superhelical plasmids upon binding, looking for changes in the linking number of the plasmid DNA. Two classical assays (the topoisomerase I-mediated relaxation assay and the ligase-mediated supercoiling assay) were performed. The results clearly indicate that the linker histones unwind superhelical DNA, with the unwinding angle being about 8° per histone H1 molecule bound to DNA. Even though this unwinding angle is small relative to the unwinding effect of other proteins, it is crucial to consider this effect in the studies of chromatin fiber structure.
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