Affiliation:
1. Oncode Institute
2. Department of Chemistry and Pharmaceutical Sciences, Amsterdam Institute of Molecular and Life Sciences (AIMMS) and Amsterdam Center for Multiscale Modeling (ACMM), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Abstract
Base-J (β-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. Α high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information.
Funder
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
KWF Kankerbestrijding
Ministerie van Onderwijs, Cultuur en Wetenschap
Publisher
Life Science Alliance, LLC
Subject
Health, Toxicology and Mutagenesis,Plant Science,Biochemistry, Genetics and Molecular Biology (miscellaneous),Ecology