Binding and dynamic studies to explore the combinatorial recognition of H3R2K9me and 5-methyl cytosine oxidation derivatives by the reader domains of UHRF1

Nivya, M Angel and Eerappa, Rajakumara (2018) Binding and dynamic studies to explore the combinatorial recognition of H3R2K9me and 5-methyl cytosine oxidation derivatives by the reader domains of UHRF1. PhD thesis, Indian institute of technology Hyderabad.

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Abstract

UHRF1 is a multi-domain protein comprising of a tandem tudor domain (UHRF1 TTD), a PHD finger, and a SET and RING-associated (UHRF1 SRA) domain. It is involved in the maintenance of CG methylation, heterochromatin formation and DNA repair processes. Isothermal titration calorimetry binding studies of UHRF1 TTD with unmodified and methylated lysine histone peptides establishes that the UHRF1 TTD binds dimethylated lysine 9 on histone H3 (H3K9me2). Further, MD simulation and binding studies reveal that, together, UHRF1’s TTD and PHD (UHRF1 TTD-PHD) preferentially recognizes dimethyllysine status. Importantly, it was found that in the binding pocket of UHRF1, the Asp145 which determines the preferential recognition of the dimethyl-ammonium group of H3K9me2. In contrast, PHD finger of the UHRF1 TTD-PHD has a negligible contribution to the binding affinity for recognition of H3K9me2 by the UHRF1 TTD-PHD. Surprisingly, Lys4 methylation on H3 peptide has an insignificant effect on combinatorial recognition of R2 and K9me2 on H3 (H3R2K9me2) by the UHRF1 TTD-PHD. Electrophoretic mobility shift assay and fluorescence polarization binding studies indicate that the UHRF1 SRA binds to all the oxidation products of 5-methyl cytosine (5mC) but exhibits lower affinity towards 5-carboxyl cytosine (5caC) and 5-formyl cytosine (5fC). Subtle variations of key residues at the binding pocket could determine status specific recognition of histone methyllysine by the reader domains. Thus, further studies are required to unravel the possibility of combinatorial recognition of H3R2K9me2, and 5hmCG DNA by the UHRF1 and role of combinatorial recognition on UHRF1 functions especially for CG methylation maintenance and heterochromatin formation.

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