D'Arpa P, Machlin P S, Ratrie H, Rothfield N F, Cleveland D W, Earnshaw W C
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543-7. doi: 10.1073/pnas.85.8.2543.
cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.
从用自身免疫抗拓扑异构酶I血清筛选的表达载体文库（λgt11）中分离出编码人拓扑异构酶I的cDNA克隆。其中一个克隆已表达为融合蛋白，该融合蛋白由细菌色氨酸E蛋白的32 kDa片段与cDNA编码的67.7 kDa蛋白相连组成。三条证据表明克隆的cDNA编码拓扑异构酶I。（i）融合蛋白和人核拓扑异构酶I的蛋白水解图谱基本相同。（ii）融合蛋白可使超螺旋DNA松弛，这种活性可被抗拓扑异构酶I血清免疫沉淀。（iii）序列分析表明，最长的cDNA克隆（3645个碱基对）编码一个765个氨基酸的蛋白，该蛋白与酿酒酵母拓扑异构酶I有42%的同源性。序列数据还表明，具有催化活性的67.7 kDa片段由羧基末端组成。
