SASDGR2: Thymine dioxygenase J-containing DNA binding domain (JDBD)

Basic information

Entry

Database: SASBDB / ID: SASDGR2

Sample

Thymine dioxygenase J-containing DNA binding domain (JDBD)

• J-DNA binding domain (protein), JDBD, Leishmania tarentolae

Function / homology

Function:

thymine dioxygenase / thymine dioxygenase activity / base J metabolic process / DNA binding / metal ion binding / nucleus

Domain/homology:

Thymine dioxygenase JBP1, DNA-binding domain / Thymine dioxygenase JBP1 DNA-binding domain / JBP1, DNA-binding domain superfamily / 2OGFeDO, oxygenase domain / Oxygenase domain of the 2OGFeDO superfamily

Component:

Thymine dioxygenase JBP1

• Biological species: Leishmania tarentolae (eukaryote)
• Citation: Journal: J Biol Chem / Year: 2019; Title: The domain architecture of the protozoan protein J-DNA-binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity.; Authors: Athanassios Adamopoulos / Tatjana Heidebrecht / Jeroen Roosendaal / Wouter G Touw / Isabelle Q Phan / Jos Beijnen / Anastassis Perrakis / ; 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 and 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 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.

Contact author

• Nassos Adamopoulos (Netherlands Cancer Institute, Amsterdam, Netherlands)

Models

Sample

• Sample: Name: Thymine dioxygenase J-containing DNA binding domain (JDBD); Specimen concentration: 3.75 mg/ml
• Buffer: Name: 20 mM HEPES, 200 mM NaCl, 1 mM TCEP / pH: 7.5

Entity #898

Name: JDBD / Type: protein / Description: J-DNA binding domain / Formula weight: 21.244 / Num. of mol.: 1 / Source: Leishmania tarentolae / References: UniProt: Q9U6M1
Sequence:

GPGPLSRLGG FSETNLMVST AVEKKKYLDS EFLLHCISAQ LLDMWKQARA RWLELVGKEW AHMLALNPER KDFLWKNQSE MNSAFFDLCE VGKQVMLGLL GKEVALPKEE QAFWIMYAVH LSAACAEELH MPEVAMSLRK LNVKLKDFNF GGTRYFKDMP PEEKKRRMER KQRIEEARRH GMP

Experimental information

• Beam: Instrument name: ESRF BM29 / City: Grenoble / 国: France / Type of source: X-ray synchrotron / Wavelength: 0.1 Å / Dist. spec. to detc.: 2.867 mm
• Detector: Name: Pilatus 1M / Type: Dectris / Pixsize x: 172 mm

Scan

Title: Thymine dioxygenase J-containing DNA binding domain (JDBD)
Measurement date: Feb 4, 2017 / Cell temperature: 4 °C / Exposure time: 1 sec. / Number of frames: 34 / Unit: 1/nm /

	Min	Max
Q	0.129	4.9417

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 881 /

	Min	Max
Q	0.218449	4.36247
P(R) point	1	881
R	0	7.1

Result

Type of curve: sec /

	Experimental	Porod
MW	28.3 kDa	24 kDa
Volume	-	38 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	7.097	7.14	0.027
Radius of gyration, Rg	2.177 nm	2.18 nm	0.03

	Min	Max
D	-	7.1
Guinier point	20	100