Title DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis /
Authors Zaremba, Mindaugas ; Toliušis, Paulius ; Grigaitis, Rokas ; Manakova, Elena ; Šilanskas, Arūnas ; Tamulaitienė, Giedrė ; Szczelkun, Mark. D ; Šikšnys, Virginijus
DOI 10.1093/nar/gku1236
Full Text Download
Is Part of Nucleic acids research.. Oxford : Oxford University Press. 2014, Vol. 42, no 22, p. 13887-13896.. ISSN 0305-1048
Abstract [eng] The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEADfamily helicase/ATPase (N.CglI and N.NgoAVII or Nproteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by Rproteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6–7 nucleotides) downstream of the asymmetric recognition sequence 5 -GCCGC-3 . Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.
Published Oxford : Oxford University Press
Type Journal article
Language English
Publication date 2014