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J Biol Chem. 2019 Aug 23;294(34):12815-12825. doi: 10.1074/jbc.RA119.007393. Epub 2019 Jul 10.

The domain architecture of the protozoan protein J-DNA-binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity.

Author information

1
Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
2
Department of Clinical Pharmacology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
3
Seattle Structural Genomics Center for Infectious Disease, Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, Washington 98109.
4
Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands a.perrakis@nki.nl.

Abstract

J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (β-d-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosoma and Leishmania JBP1 has two known functional domains: an N-terminal T hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins and a J-DNA-binding domain (JDBD) that resides in the middle of JBP1. Here, we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered structure. We found that this Δ-JDBD domain retains TH activity in vitro but displays a 15-fold lower apparent rate of hydroxylation compared with JBP1. Small-angle X-ray scattering (SAXS) experiments on JBP1 and JDBD in the presence or absence of J-DNA and on Δ-JDBD enabled us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate T hydroxylation 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module into the Δ-JDBD scaffold during evolution provided a mechanism that synergized J recognition and T hydroxylation, ensuring inheritance of base J in specific sequence patterns following DNA replication in kinetoplastid parasites.

KEYWORDS:

5-hydroxymethylcytosine (5-hmC); DNA binding protein; epigenetics; hydroxylase; kinetoplastid parasites; mass spectrometry (MS); small-angle X-ray scattering (SAXS); thymidine hydroxylase; β-D-glucosyl-hydroxymethyluracil (base J)

PMID:
31292194
PMCID:
PMC6709619
[Available on 2020-08-23]
DOI:
10.1074/jbc.RA119.007393

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