大肠杆菌DNA拓扑异构酶I对dT8及其硫代磷酸酯类似物的切割：产物与速率分析

Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis.

作者信息

Domanico P L, Tse-Dinh Y C

机构信息

Central Research and Development Department, E. I. du Pont de Nemours and Co., Wilmington, Delaware 19898.

出版信息

Biochemistry. 1988 Aug 23;27(17):6365-71. doi: 10.1021/bi00417a026.

DOI:10.1021/bi00417a026

PMID:2851323

Abstract

Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.

摘要

大肠杆菌DNA拓扑异构酶I催化以dT8作为最短可切割寡聚（胸苷酸）的短单链寡脱氧核苷酸的切割。由[5'-32P]dT8切割形成的5'-32P标记产物是dT5、dT4和dT3，超过70%的底物被切割成dT4。镁离子通过增加形成的dT4的百分比来影响这种产物分布。在磷酸二酯键中用硫原子取代非桥连氧原子会产生寡脱氧核苷酸硫代磷酸酯（PS）类似物。分离了类似物的差向异构体，并确定了硫代磷酸二酯键的位置和立体化学。拓扑异构酶I对这些类似物的反应具有立体特异性。对于寡脱氧核苷酸PS类似物底物，切割速率、立体特异性和产物分布取决于硫代磷酸二酯键的位置和立体化学。