Targeting DNA 5mCpG sites with chimeric endonucleases

Self Cite

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.

Section: Discussionmentioning

confidence: 99%

Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease

Gupta

2012

Self Cite

“…The fusion of the methyl-binding domain (MBD) of human MeCP2 (133–135) with the DNA-cleavage domain of BmrI (Class V) or FokI (Class I) had been shown to cleavage 5mCpG sequences preferentially over unmodified DNA (136). This study suggests that it is possible to generate NEases specific to 5mCpG by tethering the MBD of 5mCpG-binding proteins such as hMeCP2 and MBD2b (137–139) to an active DNA-nicking domain, such as those from Class III enzymes (i.e.…”

Section: Future Challenges and Opportunitiesmentioning

confidence: 99%

Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity

Chan¹,

Stoddard²,

Xu³

2010

238

204

Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4–8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.

“…A noteworthy exception is the fusion product consisting of a catalytically inactive homing endonuclease and the FokI cleavage domain (20), which resulted in a fusion protein recognizing the homing endonuclease recognition site and cleaving the DNA in both strands 2 and 6 nt downstream from the recognition site, as expected for the Type IIS restriction endonucleases FokI. With a similar approach the 5 mCpG-binding domain of a 5 mCpG-specific DNA glycosylase was fused to the FokI cleavage domain, in order to obtain a chimeric nuclease that cleaves DNA at 5 mCpG-sites (21). Similarly, the control protein C.BcII that represses the expression of the methyltransferase of the BcII R-M system was fused to the non-specific cleavage domain of BmrI; the chimeric nuclease recognizes specific sites in the vicinity of the C.BcII control sequence (22).…”

Section: Introductionmentioning

confidence: 99%

Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases

Fonfara

Curth

Pingoud

et al. 2011

Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.