| Title |
CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases / |
| Authors |
Toliušis, Paulius ; Zaremba, Mindaugas ; Šilanskas, Arūnas ; Szczelkun, Mark D ; Šikšnys, Virginijus |
| DOI |
10.1093/nar/gkx580 |
| Full Text |
|
| Is Part of |
Nucleic acids research.. Oxford : Oxford University Press. 2017, Vol. 45, Iss. 14, p. 8435-8447.. ISSN 0305-1048. eISSN 1362-4962 |
| Keywords [eng] |
Rrestriction endonuclease CglI ; Corynebacterium glutamicum ; NTP-hydrolysis dependent reaction ; endonuclease (R.CglI) ; DEAD-family helicase-like ATPase (H.CglI) |
| Abstract [eng] |
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. |
| Published |
Oxford : Oxford University Press |
| Type |
Journal article |
| Language |
English |
| Publication date |
2017 |
