Publication Date

April 2018

Advisor(s)

Ishita Mukerji

Major

Molecular Biology&Biochemistry

Language

English (United States)

Abstract

Separate from the core histone octamer, linker histones are known to bind to the outside of the nucleosome to regulate 30 nm chromatin fiber formation and gene expression. Unlike histone H1 found other eukaryotic organisms, the H1 linker histone homolog Hho1p, derived from Saccharomyces cerevisiae, contains two distinct globular domains (GI and GII). The individual function of each domain is unclear and it remains a question as to whether each domain plays a distinct role in nucleosomal binding. Four-way DNA junctions (4WJs) are a structural model of the naturally occurring Holliday junction, present during homologous recombination and repair. Yeast histone H1 has been shown to preferentially bind 4WJs over linear duplex DNA, in gel. We propose that H1 binding to 4WJs is structurally similar to how H1 binds to the nucleosome, where the arms of the junction resemble the DNA crossover strands of the nucleosome entry and exit site. In this project, we investigate whether S. cerevisiae histone H1 exhibits preferential binding to 4WJs over duplex DNA, in solution. Furthermore, we characterize the binding of the GII knockout, a mutant construct containing only the GI domain and N-terminal region, to both 4WJs and duplex DNA. This was conducted in order to examine how the loss of the GII domain affects the affinity of H1 for DNA. To investigate the binding affinity of yeast H1 to DNA, both wild-type S. cerevisiae H1 and the GII knockout were purified from E. coli. Binding studies reveal that wild-type H1 preferentially binds to junction over duplex DNA with a nearly 6-fold higher affinity. Specifically, we determined that wild-type H1 binds duplex with a Kd of = 4.1 nM and junction with a Kd of 0.65 nM ? 0.22 nM, in solution. Under similar conditions in our lab, mouse histone H1 has been shown to bind duplex and junction DNA with comparable affinity. Conversely, we revealed that the GII knockout does not preferentially bind 4WJs and shows a nearly 2-fold weaker affinity than wild-type H1 for 4WJs. Binding experiments in solution show that the GII knockout binds junction with a nanomolar affinity of = 1.7 nM. These results reveal that the loss of the GII domain and C-terminal region of S. cerevisiae H1 results in a diminished affinity of H1 for DNA four-way junctions. This therefore suggests that the suggests that the GI domain and N-terminal region alone are not sufficient for the high specificity binding of yeast histone H1 to DNA four-way junctions. Furthermore, both wild-type H1 and the GII knockout were determined to bind to junction with a 2:1 stoichiometry, in solution. This is different from the 1:1 stoichiometry of binding seen between canonical H1 and DNA four-way junctions.

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