Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease

Shuang Yong Xu, Yogesh K Gupta

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)378-390
Number of pages13
JournalNucleic Acids Research
Volume41
Issue number1
DOIs
StatePublished - Jan 1 2013
Externally publishedYes

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Endonucleases
Zinc Fingers
Zinc
Deoxyribonuclease I
Bacillus Phages
Binding Sites
Prophages
Molecular Pathology
Bacteriophages
Genes
Catalytic Domain
Genome
Amino Acids
DNA
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease. / Xu, Shuang Yong; Gupta, Yogesh K.

In: Nucleic Acids Research, Vol. 41, No. 1, 01.01.2013, p. 378-390.

Research output: Contribution to journalArticle

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