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Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC: evidence for a rapid conformational change upon DNA binding
Division of Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Division of Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Division of Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas, United States.
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2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 32, p. 13357-13365Article in journal (Refereed) Published
Abstract [en]

Base J (β-D-glucosyl-hydroxymethyluracil) was discovered in the nuclear DNA of some pathogenic protozoa, such as trypanosomes and Leishmania, where it replaces a fraction of base T. We have found a J-Binding Protein 1 (JBP1) in these organisms, which contains a unique J-DNA binding domain (DB-JBP1) and a thymidine hydroxylase domain involved in the first step of J biosynthesis. This hydroxylase is related to the mammalian TET enzymes that hydroxylate 5-methylcytosine in DNA. We have now studied the binding of JBP1 and DB-JBP1 to oligonucleotides containing J or glucosylated 5-hydroxymethylcytosine (glu-5-hmC) using an equilibrium fluorescence polarization assay. We find that JBP1 binds glu-5-hmC-DNA with an affinity about 40-fold lower than J-DNA (~400 nM), which is still 200 times higher than the JBP1 affinity for T-DNA. The discrimination between glu-5-hmC-DNA and T-DNA by DB-JBP1 is about 2-fold less, but enough for DB-JBP1 to be useful as a tool to isolate 5-hmC-DNA. Pre-steady state kinetic data obtained in a stopped-flow device show that the initial binding of JBP1 to glucosylated DNA is very fast with a second order rate constant of 70 μM(-1) s(-1) and that JBP1 binds to J-DNA or glu-5-hmC-DNA in a two-step reaction, in contrast to DB-JBP1, which binds in a one-step reaction. As the second (slower) step in binding is concentration independent, we infer that JBP1 undergoes a conformational change upon binding to DNA. Global analysis of pre-steady state and equilibrium binding data supports such a two-step mechanism and allowed us to determine the kinetic parameters that describe it. This notion of a conformational change is supported by small-angle neutron scattering experiments, which show that the shape of JBP1 is more elongated in complex with DNA. The conformational change upon DNA binding may allow the hydroxylase domain of JBP1 to make contact with the DNA and hydroxylate T's in spatial proximity, resulting in regional introduction of base J into the DNA.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012. Vol. 134, no 32, p. 13357-13365
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:liu:diva-143664DOI: 10.1021/ja303423tISI: 000307487200040PubMedID: 22775585Scopus ID: 2-s2.0-84865111457OAI: oai:DiVA.org:liu-143664DiVA, id: diva2:1165114
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-20Bibliographically approved

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von Castelmur, Eleonore

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