A study of SV40 protein-nucleic acid interactions and sequence-dependent protein-induced DNA bending

Henry Bernard Lowman, Purdue University

Abstract

SV40 is used as a model system to study eukaryotic chromatin because the viral genomic DNA form a minichromosome, with packing features much like those of higher eukaryotes. Broadly speaking, SV40 DNA undergoes interactions with three clauses of proteins: the host-derived histones, proteins encoded by the SV40 genome itself, and regulatory proteins of the host. To study the interactions of DNA with two of these classes of protein, experimental and theoretical approaches were taken to examine the interactions of core histones with SV40 DNA in vivo, and expression systems were developed for the biosynthesis of SV40 proteins in E. coli as a starting point for in vitro experiments. Mouse satellite nucleosomal DNA was first used as a model for examining DNA sequence features common to nucleosomes on a highly repetitive sequence. The interactions of the core histones with SV40 DNA were examined by determining the placement of nucleosomes on the genome through the use of a shotgun-cloning technique. These nucleosome locations were used to generate a set of core-length nucleosomal DNA sequences which were compared with models for sequence-dependent variations in DNA structure. The results indicate that certain regions of the genome favor nucleosome placement, with a few "strong" sites being represented significantly more than average. However, it appears that SV40 nucleosome placement does not follow strict positioning and that simple dinucleotide periodicities are not common among all nucleosome sites, nor among the strongest sites. It therefore seems that a number of different arrangements of nucleosomes on the genome are possible and that factors other than the DNA sequence must contribute to nucleosome placement on SV40 chromatin. One protein which has been previously implicated in interactions with SV40 chromatin is the SV40 agnoprotein, or LP1. Studies to examine the interactions between LP1 and SV40 DNA have been initiated by developing an expression system for the biosynthesis of a heterologous protein, NS1A-LP1, which appears to bind supercoiled DNA in preference to double stranded DNA, and which may be useful for future studies of how non-histone proteins contribute to the organization of SV40 chromatin structure.

Degree

Ph.D.

Advisors

Bina, Purdue University.

Subject Area

Biochemistry

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