6GHC

Modification dependent EcoKMcrA restriction endonuclease

Summary for 6GHC

• Entry DOI: 10.2210/pdb6ghc/pdb
• Descriptor: 5-methylcytosine-specific restriction enzyme A, ZINC ION (3 entities in total)
• Functional Keywords: hnh endonuclease, modification dependent restriction, 5-methylcytosine, 5mc, 5-hydroxymethylcytosine, 5hmc, bba-me nuclease, scomcra, hydrolase
• Biological source: Escherichia coli (strain K12)
• Total number of polymer chains: 2
• Total formula weight: 65714.03

Authors

Czapinska, H.,Kowalska, M.,Zagorskaite, E.,Manakova, E.,Xu, S.,Siksnys, V.,Sasnauskas, G.,Bochtler, M. (deposition date: 2018-05-07, release date: 2018-08-08, Last modification date: 2024-05-15)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2.85 Å)