Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change

Joanna Żebrowska , Joanna Jeżewska-Frąckowiak , Ewa Wieczerzak , Franciszek Kasprzykowski , Agnieszka Żylicz-Stachula , Piotr Skowron


Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4–8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit ‘affinity star’ activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in ‘star’ activity, exemplified by TsoI. The TsoI ‘star’ activity cannot be described under the definition of the classic ‘star’ activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.
Author Joanna Żebrowska (FCh / DMBt / LGE)
Joanna Żebrowska,,
- Laboratory of Genetic Engineering
, Joanna Jeżewska-Frąckowiak (FCh / DMBt / LGE)
Joanna Jeżewska-Frąckowiak,,
- Laboratory of Genetic Engineering
, Ewa Wieczerzak (FCh / DBCh / LMCh)
Ewa Wieczerzak,,
- Laboratory of Medical Chemistry
, Franciszek Kasprzykowski (FCh / DBCh / LMCh)
Franciszek Kasprzykowski,,
- Laboratory of Medical Chemistry
, Agnieszka Żylicz-Stachula (FCh / DMBt / LGE)
Agnieszka Żylicz-Stachula,,
- Laboratory of Genetic Engineering
, Piotr Skowron (FCh / DMBt / LGE)
Piotr Skowron,,
- Laboratory of Genetic Engineering
Journal seriesApplied Microbiology and Biotechnology, ISSN 0175-7598, (N/A 100 pkt)
Issue year2019
Publication size in sheets0.6
Keywords in EnglishRestriction endonucleases, DNA recognition specificity 'Star' activity, S-adenosyl-L-cysteine, DNA fragmentation
ASJC Classification2700 General Medicine; 1305 Biotechnology; 2402 Applied Microbiology and Biotechnology
Languageen angielski
LicenseOther; published final; Uznanie Autorstwa (CC-BY); with publication
Score (nominal)100
Score sourcejournalList
ScoreMinisterial score = 100.0, 28-01-2020, ArticleFromJournal
Publication indicators WoS Citations = 0; Scopus SNIP (Source Normalised Impact per Paper): 2018 = 1.236; WoS Impact Factor: 2018 = 3.67 (2) - 2018=3.889 (5)
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