Talk abstracts

Talk on Thursday 05:00-05:15pm submitted by Sarah Javaid

Nucleosome Remodeling by hMSH2-hMSH6

Sarah Javaid (Biophysics Program, Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University), Nidhi Punja (Department of Molecular Virology, Immunology, and Medical Genetics; Human Cancer Genetics and The Ohio State University), Alex Mooney (Department of Physics and The Ohio State University), Mridula Manohar, Jennifer Ottesen (Department of Biochemistry and The Ohio State University), Michael Poirier (Departments of Molecular Virology, Immunology, and Medical Genetics; Human Cancer Genetics, Biochemistry, and Physics and The Ohio State University), Richard Fishel (Departments of Molecular Virology, Immunology, and Medical Genetics; Human Cancer Genetics, and Physics and The Ohio State University)

Abstract:
The human MutS homologs (MSH), hMSH2 and hMSH6, form a heterodimer (hMSH2-hMSH6) that plays a central role in mismatch repair (MMR). hMSH2-hMSH6 recognize mismatched nucleotides generated by misincorporation during DNA replication as well as lesions generated by spontaneous and chemical-induced DNA damage. Mutations in the hMSH2 or hMSH6 genes result in susceptibility to the common cancer predisposition syndrome, Hereditary Non-Polyposis Colorectal Cancer (HNPCC).

Mismatches and lesions that are recognized by hMSH2-hMSH6 arise within chromosomes that are a complex mixture of DNA and protein (chromatin). The fundamental unit of chromatin is the nucleosome which consists of ~146 bp of DNA wrapped twice around a histone octomer containing two H2A-H2B dimers and an H3-H4 tetramer. We have been examining the biophysical effect of chromatin on MMR processes, as well as the effect of post-translational modifications that decorate the human histones.

We have developed a mismatched DNA substrate containing a single well-defined nucleosome. We demonstrate that hMSH2-hMSH6 can catalyze the displacement of a nucleosome adjacent to a mismatch. In addition, we have constructed nucleosomes containing acetylations (ac) of the histone H3(K115), H3(K122), and H3(K56) by a semi-synthetic intein-based strategy. We find that hMSH2-hMSH6 displacement is considerably enhanced when nucleosomes contain H3(K115ac), H3(K122ac), and H3(K56ac). Lys->Gln substitution mutations of H3(K115), H3(K122), and H3(K56), which are used to mimic Lys acetylations, also enhance nucleosome displacement. We find that hMSH2-hMSH6 nucleosome displacement is dependent on the Nucleosome Positioning Sequence (NPS). hMSH2-hMSH6 nucleosome displacement is enhanced with the 5S rDNA positioning sequence whereas the displacement of the nucleosome is significantly inhibited by the nonphysiological 601 and MP2 positioning sequences. hMSH2-hMSH6 also catalyzes the displacement of nucleosomes which contain a mismatch within the 5S rDNA NPS. This displacement is enhanced when the nucleosomes contain H3(K115ac,K122ac) compared to unmodified nucleosomes. These results highlight that nucleosome displacement by hMSH2-hMSH6 is highly dependent on the NPS, post-translational modifications, and/or mimics.

Keywords: Mismatch Repair, Chromatin, Cancer