DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis

Nucleic Acids Res. 2014 Dec 16;42(22):13887-96. doi: 10.1093/nar/gku1236. Epub 2014 Nov 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Corynebacterium glutamicum / enzymology
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / isolation & purification
  • DEAD-box RNA Helicases / metabolism*
  • DNA / metabolism
  • DNA Cleavage*
  • DNA Restriction Enzymes / chemistry
  • DNA Restriction Enzymes / isolation & purification
  • DNA Restriction Enzymes / metabolism*
  • Hydrolysis
  • Neisseria gonorrhoeae / enzymology
  • Nucleotides / metabolism
  • Protein Structure, Tertiary

Substances

  • Nucleotides
  • Adenosine Triphosphate
  • DNA
  • DNA Restriction Enzymes
  • DEAD-box RNA Helicases