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Abstract

Abstract: Restriction endonucleases are essential tools in genetic engineering, cloning and DNA diagnostics. N.BstNBI is a natural occurring nicking endonuclease which only breaks one strand of double-stranded DNA, and it is the only nicking enzyme that is commercially available. The proposed research is about engineering Type IIs endonucleases into nicking enzymes. In Phase I research, we have cloned and characterized the nicking endonuclease N.BstNBI and two related type IIs endonucleases, PleI and MlyI. Our results showed that type IIs endonucleases PleI and MlyI carry out double-stranded cleavage in a two-step process, in which the DNA is first nicked and then further cleaved on second strand by another enzyme molecule via dimerization. In the case of N.BstNBI, it is the dimerization function that was probably affected by mutation, resulting in a unique endonuclease which nicks DNA. We have successfully converted MlyI into a nicking endonuclease by disrupting its dimerization function. Thus, we have demonstrated that it is possible to convert Type IIs endonucleases into nicking enzymes by mutations. We will apply the techniques used in Phase I as well as additional strategies to convert more Type IIs endonucleases into new nicking enzymes. PROPOSED COMMERCIAL APPLICATIONS: Potentially can lead to the conversion of type IIs restriction endonucleases into nicking enzymes which have great applications in DNA amplification technology (SDA, RCA) and research (DNA replication, repair, etc.).

Thesaurus Terms:
enzyme activity, nucleic acid sequence, protein engineering, restriction endonuclease
DNA topoisomerase, active site, gene expression, genetic manipulation, molecular cloning
plasmid, site directed mutagenesis

Institution:	NEW ENGLAND BIOLABS, INC.
	32 TOZER RD
	BEVERLY, MA 01915
Fiscal Year:	2002
Department:
Project Start:	01-JUL-1999
Project End:	31-AUG-2003
ICD:	NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG:	ZRG1