title: DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis.
abstract: 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 DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). 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 R-proteins. 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.
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania zare@ibt.lt.
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Zaremba
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Paulius
authors.initials: P
authors.lastname: Toliusis
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Rokas
authors.initials: R
authors.lastname: Grigaitis
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Elena
authors.initials: E
authors.lastname: Manakova
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Arunas
authors.initials: A
authors.lastname: Silanskas
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Giedre
authors.initials: G
authors.lastname: Tamulaitiene
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania siksnys@ibt.lt.
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D009344:Neisseria gonorrhoeae / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D009711:Nucleotides; D000255:Adenosine Triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; D053487:DEAD-box RNA Helicases