Degenerate sequence recognition by the monomeric restriction enzyme: single mutation converts BcnI into a strand-specific nicking endonuclease

Kostiuk, Georgij; Sasnauskas, Giedrius; Tamulaitiene, G.; Šikšnys, Virginijus

doi:10.1093/nar/gkq1351

Cited by 14 publications

(17 citation statements)

References 34 publications

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“…Site-directed mutagenesis of the two His residues generated two nicking variants, H77A and H219Q, that prefer to nick 5′-CCCGG-3′ and 5′-GGGCC-3′, respectively. Major DNA nicking product by H219Q was detected in a short digestion, but longer digestion also generated some dsDNA cleavage products (56). It was also feasible to lower the pH in digestion buffer to force BfiI to nick only the bottom strand in 5′-ACTGGG-3′ (57).…”

Section: Dna Recognition Site Mutation or Cofactor (Ph) Alterationmentioning

confidence: 99%

Sequence-specific DNA nicking endonucleases

2015

Biomolecular Concepts

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A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma. A minimal nicking domain of 76 amino acid residues from N.ϕGamma could be fused to other DNA binding partners to generate chimeric NEases with new specificities. The biological roles of a few small HNH endonucleases (HNHE, gp74 of HK97, gp37 of ϕSLT, ϕ12 HNHE) have been demonstrated in phage and pathogenicity island DNA packaging. Another group of NEases with 3- to 7-bp specificities are either natural components of restriction systems or engineered from type IIS restriction endonucleases. A phage group I intron-encoded HNH homing endonucleases, I-PfoP3I was found to nick DNA sites of 14-16 bp. I-TslI encoded by T7-like ΦI appeared to nick DNA sites with a 9-bp core sequence. DNA nicking and labeling have been applied to optical mapping to aid genome sequence assembly and detection of large insertion/deletion mutations in genomic DNA of cancer cells. Nicking enzyme-mediated amplification reaction has been applied to rapid diagnostic testing of influenza A and B in clinical setting and for construction of DNA-based Boolean logic gates. The clustered regularly interspaced short palindromic repeats-ribonucleoprotein complex consisting of engineered Cas9 nickases in conjunction with tracerRNA:crRNA or a single-guide RNA have been successfully used in genome modifications.

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Section: Dna Recognition Site Mutation or Cofactor (Ph) Alterationmentioning

confidence: 99%

Sequence-specific DNA nicking endonucleases

2015

Biomolecular Concepts

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“…In contrast, relatively few natural nicking endonucleases (NEases or nickase), which introduce single-strand breaks in double-stranded DNA (dsDNA), have been discovered (3,6–8). NEases were engineered from type IIS REases by mutating one of the two catalytic sites or disrupting the dimerization domain (9–11), by alteration of binding specificity of a type IIP REase (12) or by combining a DNA binding-deficient and catalytic-proficient FokI subunit with a DNA binding-proficient and catalytically inert FokI subunit (13). Naturally occurring nicking enzymes may be found as part of heterodimeric REases or as stand-alone enzymes.…”

Section: Introductionmentioning

confidence: 99%

Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease

Gupta

2012

Nucleic Acids Research

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Many bacteriophage and prophage genomes encode an HNH endonuclease (HNHE) next to their cohesive end site and terminase genes. The HNH catalytic domain contains the conserved catalytic residues His-Asn-His and a zinc-binding site [CxxC]2. An additional zinc ribbon (ZR) domain with one to two zinc-binding sites ([CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC] or [CxxH]) is frequently found at the N-terminus or C-terminus of the HNHE or a ZR domain protein (ZRP) located adjacent to the HNHE. We expressed and purified 10 such HNHEs and characterized their cleavage sites. These HNHEs are site-specific and strand-specific nicking endonucleases (NEase or nickase) with 3- to 7-bp specificities. A minimal HNH nicking domain of 76 amino acid residues was identified from Bacillus phage γ HNHE and subsequently fused to a zinc finger protein to generate a chimeric NEase with a new specificity (12–13 bp). The identification of a large pool of previously unknown natural NEases and engineered NEases provides more ‘tools’ for DNA manipulation and molecular diagnostics. The small modular HNH nicking domain can be used to generate rare NEases applicable to targeted genome editing. In addition, the engineered ZF nickase is useful for evaluation of off-target sites in vitro before performing cell-based gene modification.

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“…Finally, BcnI (5′-CC/SGG-3′), and the closely related REase MvaI (5′-CC/WGG-3′) are monomeric one-domain enzymes containing a single catalytic center (Figure 1(D) ( 11 , 12 )). To accomplish cleavage of double-stranded DNA these enzymes must first bind the recognition sequence in one orientation, nick the first DNA strand, then re-bind the nicked recognition site in the opposite orientation and nick the second DNA strand ( 13 ). Surprisingly, biochemical studies of BcnI and preliminary characterization of MvaI suggest that both these enzymes, despite having a single catalytic center, are highly efficient REases, capable of cleaving both strands of the recognition site during a single binding event ( 14 ).…”

Section: Introductionmentioning

confidence: 99%

UbaLAI is a monomeric Type IIE restriction enzyme

Sasnauskas

Tamulaitiene

Тамулайтис

et al. 2017

Nucleic Acids Research

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Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5′-CC/WGG-3′ (where W = A/T, and ‘/’ marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5′-CCWGG-3′ sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5′-CCWGG-3′ sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.

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Degenerate sequence recognition by the monomeric restriction enzyme: single mutation converts BcnI into a strand-specific nicking endonuclease

Cited by 14 publications

References 34 publications

Sequence-specific DNA nicking endonucleases

Sequence-specific DNA nicking endonucleases

Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease

UbaLAI is a monomeric Type IIE restriction enzyme

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