ALTERATION OF THE CLEAVAGE DISTANCE OF FOK-I RESTRICTION ENDONUCLEASE BY INSERTION MUTAGENESIS

被引：42

作者：

LI, L ^{[1
]}

CHANDRASEGARAN, S ^{[1
]}

机构：

[1] JOHNS HOPKINS UNIV,SCH HYG & PUBL HLTH,DEPT ENVIRONM HLTH SCI,DIV ENVIRONM CHEM & BIOL,BALTIMORE,MD 21205

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1993年 / 90卷 / 07期

关键词：

FLAVOBACTERIUM-OKEANOKOITIES; ESCHERICHIA-COLI; RECOGNITION AND CLEAVAGE DOMAINS; PROTEIN ENGINEERING;

D O I：

10.1073/pnas.90.7.2764

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'-5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site. Recently, we reported the presence of two distinct and separable protein domains within this enzyme-one for the sequence-specific recognition and the other for endonuclease activity. Here, we report the construction of two insertion mutants of Fok I endonuclease. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence specificity as the wild-type enzyme. However, compared with the wild-type enzyme, they cleave one nucleotide further away from the recognition site on both strands of the DNA substrates. Thus, it is possible to alter the cleavage distance of Fok I by protein engineering.

引用

页码：2764 / 2768

页数：5

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