Investigation into the Binding Interactions of Saccharomyces cerevisiae Histone H1 with Holliday Junction.

Abstract

In eukaryotic organisms, DNA is tightly packaged into chromatin through association with proteins to form a highly condensed structure called the chromosome. Linker histone H1 binds DNA at the nucleosome entry and exit points and potentially regulates chromosome structure and gene expression. Unlike histone H1 found in many higher order eukaryotic organisms, which contain only one globular domain, Saccharomyces cerevisiae (S. cerevisiae) histone H1 contains two distinct globular domains. The importance of the two individual globular domains with respect to binding and recognition remains unclear. Four‐way DNA junctions (4WJs) are a structural model of the naturally occurring Holliday junction present during homologous recombination and repair, and H1 has previously been shown to bind 4WJs preferentially. We propose that H1 binding to 4WJs is similar structurally 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. We are investigating whether yeast histone H1 exhibits preferential binding to 4WJ over that of duplex DNA. We are also examining how the presence of the two globular domains affects H1 binding to both 4WJs and duplex DNA.To investigate the binding affinity of yeast H1 to DNA, S. cerevisiae H1 was purified from an E. coli strain overexpressing the protein and characterized through DNA binding assays. Although H1 has previously been shown to preferentially bind to junction over duplex DNA, preliminary results of our DNA binding studies suggest that H1 shows only a slight preference for junction DNA. Results of gel mobility shift assays (GMSA) show that H1 binds to duplex DNA with a KD of 33.6 nM, and junction with a KD of 24.5 nM. Fluorescence intensity experiments are relatively consistent with GMSA results. Specifically, we determined that H1 binds duplex with a KD of 18.8 ± 2.1 nM and junction with a KD of 7.7 ± 1.1 nM. These solution results suggest that H1 has a 2–3 fold higher affinity for the 4WJ relative to duplex DNA. Under similar solution conditions, mouse histone H1 binds to duplex and junction DNA with comparable affinity. Therefore, our current results suggest that the binding of yeast H1 to both junction and duplex is similar to mouse histone H1, despite the presence of its second globular domain.The effect of the two globular domains of S. cerevisiae H1 on DNA binding was further investigated through the creation of two mutant strains, each containing only one of the two globular domains. These mutants have been overexpressed and purified from E. Coli to examine the differential binding properties of the individual domains. The binding affinities of these constructs for junction and duplex DNA will be presented.Support or Funding InformationThis work was supported by the ASBMB Summer Undergraduate Research Award and the National Science Foundation (MCB‐0843656)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.