Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I

Krefft, Daria; Żylicz-Stachula, Agnieszka; Mulkiewicz, Ewa; Papkov, Aliaksei; Jeżewska-Frąckowiak, Joanna; Skowron, Piotr M.

doi:10.1016/j.jbiotec.2014.11.030

Cited by 10 publications

(29 citation statements)

References 28 publications

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“…The ‘affinity star’ activity induced by the SAM analogue was previously observed for two enzymes from the Tsp GWI subfamily: Tsp GWI and Taq II (Zylicz-Stachula et al 2009 , 2011b , 2013 ) and one enzyme from the Tsp DTI subfamily: Tth HB27I (Krefft et al 2018 ). Here, we have shown that a similar phenomenon could also be triggered for Tso I - another enzyme from the Tsp DTI subfamily (Skowron et al 2003 , 2013 ; Zylicz-Stachula et al 2012 ; Krefft et al 2015 ). This effect is related to the previously defined ‘affinity star’ activity (Zylicz-Stachula et al 2011b , 2013 ) as it can be enhanced by the addition of SAH analogue to the reaction buffer.…”

Section: Discussionsupporting

confidence: 55%

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

Żebrowska

Jeżewska-Frąckowiak

Wieczerzak

et al. 2019

Appl Microbiol Biotechnol

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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. Tso I 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 Tso I. The Tso I ‘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 Tso I SCS comprises several degenerated variants of the cognate site. Although the efficiency of Tso I SCS cleavage is lower in comparison to the cognate Tso I recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The Tso I/SAC REase may serve as a novel tool for DNA manipulation. Electronic supplementary material The online version of this article (10.1007/s00253-019-09731-0) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 55%

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

Żebrowska

Jeżewska-Frąckowiak

Wieczerzak

et al. 2019

Appl Microbiol Biotechnol

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“…2a, b , all 5′-CCNGG-3′ are cleaved both by MmoSTI and the prototype ScrFI. To finally validate this analysis, a comparative digestion of the pUC19 plasmid, as well as a long 1789 bp PCR substrate (Krefft et al 2015 ), with MmoSTI and ScrFI was conducted and resulted in the same digestion patterns. As both enzymes cleave DNA frequently, the obtained multiple bands are grouped within a small fragment size range (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 and 2 ). The 390 bp substrate was amplified with sequence-modifying primers from a pBR322 template ( Zylicz-Stachula et al 2011 ) and the 1789 bp substrate was amplified from the pACYC184 plasmid (Krefft et al 2015 ). Four variants of the 54 bp substrate, differing in a single bp within isolated 5′-CCNGG-3′ sites, were prepared as follows: four 54 nt ssDNAs were synthesized and used as a PCR template (MmoSTI putative recognition sites in bold, underlined): 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT CCAGG GCCGCGCTGCGGACCC-3′, 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT CCTGG GCCGCGCTGCGGACCC-3′, 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT CCCGG GCCGCGCTGCGGACCC-3′, 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT CCGGG GCCGCGCTGCGGACCC-3′.…”

Section: Methodsmentioning

confidence: 99%

MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5′-|CCNGG-3′ sequences

et al. 2015

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A type II restriction endonuclease, MmoSTI, from the pathogenic bacterium Morganella morganii infecting a tropical moth, Actias selene, has been detected and biochemically characterized, as a potential etiological differentiation factor. The described REase recognizes interrupted palindromes, i.e., 5′-CCNGG-3′ sequences and cleaves DNA leaving 5-nucleotide (nt) long, single-stranded (ss), 5′-cohesive ends, which was determined by three complementary methods: (i) cleavage of custom and standard DNA substrates, (ii) run-off sequencing of cleavage products, and (iii) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with MmoSTI. MmoSTI, the first 5′-CCNGG-3′ REase characterized from M. morganii, is a neoschizomer of ScrFI, which cleaves DNA leaving 1-nt long, ss, 5′-cohesive ends. It is a high-frequency cutter and can be isolated from easily cultured bacteria, thus it can potentially serve as a tool for DNA manipulations.

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“…To accumulate possible problems to test within a single target, we selected a very toxic gene–coding for DNA cleaving REase-MTase RM.TthHB27I, which originates from a high GC content thermophile. RM.TthHB27I protein is a member of the Thermus- family of atypical, bifunctional thermozymes, which was characterized by our group [ 21 – 24 ]. The selected Type IIC/IIG/IIS REase-MTase recognizes asymmetric 5’-CAARCA-3’ DNA sequences and cleaves 11/9 nt downstream [ 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene

et al. 2017

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Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.

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Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I

Cited by 10 publications

References 28 publications

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

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

MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5′-|CCNGG-3′ sequences

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene

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