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Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.

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journal contribution
posted on 2012-10-24, 09:13 authored by G. Liu, H. Y. Ou, T. Wang, L. Li, H. Tan, X. Zhou, Kumar Rajakumar, Z. Deng, X. He
Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.

Funding

This work was supported by Grants 973 and 863 from the Ministry of Science and Technology, the National Science Foundation of China, The Royal Society-NSFC International Joint Program (2007/R3) to KR and ZD, the Ministry of Education, the Science and Technology Commission of Shanghai Municipality, and Shanghai Leading Academic Discipline Project B203.

History

Citation

PLoS Genetics, 2010, 6 (12), pp. e1001253-e1001253

Published in

PLoS Genetics

Publisher

Public Library of Science

issn

1553-7390

eissn

1553-7404

Copyright date

2010

Available date

2012-10-24

Publisher version

http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1001253

Language

eng