報告題目：Strand-specific and sequence-specific DNA nicking enzymes

報  告 人：Shuang-yong Xu, Senior Scientist

New England Biolabs, Ipswich, MA, USA.

報告時間🤭：7月28日  上午9🤦‍♀️：30-11🚣🏽‍♀️：00

報告地點: 徐匯校區哲生館一樓會議室

聯 系 人𓀏：賀新義 13651667745

報告簡介:

Dr. Xu’s lab cloned and expressed natural nicking enzymes N.CviPII, N.CviQII, Nb.BsrDI and Nb.BtsI. Recently his lab co-discovered a HNH family nicking endonucleases that are encoded by phage and prophage. One HNH endonuclease, N.ϕGamma, was characterized and shown to nick DNA at ACCGR. Under star condition, the nicking specificity can be relaxed to ACCG. A minimal DNA nicking domain of 76-aa was defined from N.ϕGamma by deletion analysis. The minimal nicking domain was fused to zinc finger protein or cleavage-deficient BamHI to generate rare nicking enzymes. The nicking domain was also fused to MBD to generate 5mCG targeting nicking enzyme. By protein engineering, Dr. Xu’s lab also generated nicking variants from BspQI, BsmI, BsmAI, and BtsCI. The nicking enzymes are useful tools to nick and label DNA for optical mapping (nicking site profile) for diagnostic applications and genome contig assembly. In other enzyme engineering efforts, Dr. Xu lab was also involved in engineering high fidelity restriction enzymes such as KpnI-HF and BamHI-HF. Most recently, his lab cloned and characterized 26 active BisI family homologs that cleave GCNGC sites with 2-4 modified 5mC.