Cal S, Tan K L, McGregor A, Connolly B A
Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
EMBO J. 1998 Dec 1;17(23):7128-38. doi: 10.1093/emboj/17.23.7128.
Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.
牛胰脱氧核糖核酸酶I（DNase I）是一种特异性相对较低的核酸酶，它在小沟中与DNA相互作用。蛋白质与核酸的大沟之间没有接触。DNase I在结构上与核酸外切酶III同源，核酸外切酶III是一种具有多种活性的DNA修复酶。这两种酶的主要区别之一是核酸外切酶III中存在一个额外的α螺旋，其位置表明与DNA的大沟相互作用。重组DNA技术已被用于向DNase I添加14个氨基酸，包括核酸外切酶IIIα螺旋的10个氨基酸，两侧是富含甘氨酸的区域。该多肽已插入到丝氨酸174之后，丝氨酸174是DNase I表面的一个氨基酸，与核酸外切酶III中额外α螺旋的位置相对应。重组蛋白DNase-exohelix已被纯化，并对其对DNA的催化活性进行了研究。重组蛋白对DNA中无碱基位点的内切核酸酶切割表现出高选择性，这是核酸外切酶III的特性，而天然DNase I则没有。因此，在Ser174处插入14个氨基酸，可将DNase I转化为具有DNA修复特性的类似核酸外切酶III的酶。
