Analysis of the protein-DNA interactions of nucleosomes containing SV40 DNA
Abstract
In order to enhance our understanding of the potential effects of nucleosome assembly on chromatin function and to examine the effects of DNA structure on nucleosome formation, in vitro reconstitution was performed using two short DNA segments selected from the simian virus 40 (SV40) genome. Hydroxyl radical and DNase I footprinting techniques were applied to examine the structure of the reconstituted nucleosomes. One fragment included the strongest nucleosome location identified by shot-gun cloning analysis of SV40 nucleosomal fragments. This site is near a potential late boundary for the nuclease-hypersensitive region in SV40 chromatin. In vitro constitution analysis enabled us to determine whether the underlying DNA sequence encodes for the assembly of this nucleosome. Both footprinting reagents revealed the formation of a precisely positioned nucleosome in vitro, whose center maps between nucleotide 384 and 387 on SV40 DNA. The corresponding nucleosome core includes the major-late transcription initiation site (12 bps within the core), the MspI site, and a segment shown previously to contain bent DNA in the absence of proteins. The dominant periodicity of DNA in this nucleosome is 10.26 bps per turn. The other fragment used in our studies contained part of the SV40 Hind II + III F fragment which was reconstituted with core histones in order to examine how a DNA segment containing a small sharply bent region (44 bps) would interact with core histones. Footprinting analysis detected the formation of a precisely positioned nucleosome which encompassed nt 1819-1966 on SV40 DNA. The bent region is located adjacent to the dyad (nt 1892.5) of the nucleosome formed. The dominant periodicity of DNA in this nucleosome is 10.3 bps per turn for the noncoding and 10.4 bps per turn for the coding strand. In both nucleosomes, the tracts of A's attributed to DNA bending adopts a defined rotational setting. Overall, the distribution of the OH cut sites provides strong support for models in which the minor grooves of the A/T-rich tracts are oriented toward the histone core while the minor grooves of the G/C-rich sequences are facing outward.
Degree
Ph.D.
Advisors
Bina, Purdue University.
Subject Area
Biochemistry
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