SASDGS2: J-23-DNA (J-DNA (23mer))

基本情報

登録情報

データベース: SASBDB / ID: SASDGS2

試料

J-23-DNA

• J-DNA (23mer) (DNA), J-23-DNA

• 引用: ジャーナル: J Biol Chem / 年: 2019; タイトル: The domain architecture of the protozoan protein J-DNA-binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity.; 著者: Athanassios Adamopoulos / Tatjana Heidebrecht / Jeroen Roosendaal / Wouter G Touw / Isabelle Q Phan / Jos Beijnen / Anastassis Perrakis / ; 要旨: 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.

登録者

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

モデル

試料

• 試料: 名称: J-23-DNA / 試料濃度: 4.1 mg/ml
• バッファ: 名称: 20 mM HEPES, 200 mM NaCl, 1 mM TCEP / pH: 7.5

要素 #899

名称: J-23-DNA / タイプ: DNA / 記述: J-DNA (23mer) / 分子量: 14.225 / 分子数: 1
配列:

TCGATTTGTT CATAGACTAA TACAGCTAAA CAAGTATCTG ATTATG

実験情報

• ビーム: 設備名称: ESRF BM29 / 地域: Grenoble / 国: France / 線源: X-ray synchrotron / 波長: 0.1 Å / スペクトロメータ・検出器間距離: 2.872 mm
• 検出器: 名称: Pilatus 1M / タイプ: Dectris / Pixsize x: 172 mm

スキャン

タイトル: J-23-DNA / 測定日: 2016年2月21日 / セル温度: 4 °C / 照射時間: 1 sec. / フレーム数: 31 / 単位: 1/nm /

	Min	Max
Q	0.1036	4.9448

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 899 /

	Min	Max
Q	0.108292	4.34138
P(R) point	1	899
R	0	7.3

結果

カーブのタイプ: sec /

	実験値	Porod
分子量	16.2 kDa	-
体積	-	20 nm3

	P(R)	Guinier	Guinier error
前方散乱 I0	4.557	4.53	0.012
慣性半径, Rg	2.34 nm	2.26 nm	0.01

	Min	Max
D	-	7.3
Guinier point	2	100