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

Mindaugas Zaremba, Paulius Toliusis, Rokas Grigaitis, Elena Manakova, Arunas Silanskas, Giedre Tamulaitiene, Mark D Szczelkun, Virginijus Siksnys

Research output: Contribution to journalArticle (Academic Journal)peer-review

8 Citations (Scopus)
388 Downloads (Pure)

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.

Original languageEnglish
Pages (from-to)13887-13896
Number of pages10
JournalNucleic Acids Research
Volume42
Issue number22
Early online date27 Nov 2014
DOIs
Publication statusPublished - 16 Dec 2014

Fingerprint

Dive into the research topics of 'DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis'. Together they form a unique fingerprint.

Cite this