CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases

Paulius Toliusis, Mindaugas Zaremba, Arunas Silanskas, Mark D Szczelkun, Virginijus Siksnys

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

2 Citations (Scopus)
442 Downloads (Pure)

Abstract

The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes.
Original languageEnglish
Pages (from-to)8435-8447
Number of pages13
JournalNucleic Acids Research
Volume45
Issue number14
Early online date7 Jul 2017
DOIs
Publication statusPublished - 21 Aug 2017

Keywords

  • DNA
  • DNA restriction enzymes
  • translocation (genetics)
  • enzymes
  • cytokinesis
  • DNA cleavage

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