6GHC

Modification dependent EcoKMcrA restriction endonuclease

6GHC の概要

• エントリーDOI: 10.2210/pdb6ghc/pdb
• 分子名称: 5-methylcytosine-specific restriction enzyme A, ZINC ION (3 entities in total)
• 機能のキーワード: hnh endonuclease, modification dependent restriction, 5-methylcytosine, 5mc, 5-hydroxymethylcytosine, 5hmc, bba-me nuclease, scomcra, hydrolase
• 由来する生物種: Escherichia coli (strain K12)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 65714.03

構造登録者

Czapinska, H.,Kowalska, M.,Zagorskaite, E.,Manakova, E.,Xu, S.,Siksnys, V.,Sasnauskas, G.,Bochtler, M. (登録日: 2018-05-07, 公開日: 2018-08-08, 最終更新日: 2024-05-15)

主引用文献

Czapinska, H.,Kowalska, M.,Zagorskaite, E.,Manakova, E.,Slyvka, A.,Xu, S.Y.,Siksnys, V.,Sasnauskas, G.,Bochtler, M.
Activity and structure of EcoKMcrA.
Nucleic Acids Res., 46:9829-9841, 2018

PubMed Abstract: Escherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of EcoKMcrA that includes the first demonstration of its endonuclease activity, small angle X-ray scattering (SAXS) data, and a crystal structure of the enzyme in the absence of DNA. Our data indicate that EcoKMcrA dimerizes via the anticipated C-terminal HNH domains, which together form a single DNA binding site. The N-terminal domains are not homologous to SRA domains, do not interact with each other, and have separate DNA binding sites. Electrophoretic mobility shift assay (EMSA) and footprinting experiments suggest that the N-terminal domains can sense the presence and sequence context of modified cytosines. Pyrrolocytosine fluorescence data indicate no base flipping. In vitro, EcoKMcrA DNA endonuclease activity requires Mn2+ ions, is not strictly methyl dependent, and is not observed when active site variants of the enzyme are used. In cells, EcoKMcrA specifically restricts DNA that is modified in the correct sequence context. This activity is impaired by mutations of the nuclease active site, unless the enzyme is highly overexpressed.

PubMed: 30107581
DOI: 10.1093/nar/gky731

実験手法

X-RAY DIFFRACTION (2.85 Å)