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 language | English (US) |
|---|---|
| Pages (from-to) | 378-390 |
| Number of pages | 13 |
| Journal | Nucleic Acids Research |
| Volume | 41 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2013 |
| Externally published | Yes |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease
AU - Xu, Shuang Yong
AU - Gupta, Yogesh K
PY - 2013/1/1
Y1 - 2013/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84871802688&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84871802688&partnerID=8YFLogxK
U2 - 10.1093/nar/gks1043
DO - 10.1093/nar/gks1043
M3 - Article
C2 - 23125367
AN - SCOPUS:84871802688
VL - 41
SP - 378
EP - 390
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 1
ER -