CDNA CLONING OF HUMAN DNA TOPOISOMERASE-I - CATALYTIC ACTIVITY OF A 67.7-KDA CARBOXYL-TERMINAL FRAGMENT
CDNA CLONING OF HUMAN DNA TOPOISOMERASE-I - CATALYTIC ACTIVITY OF A 67.7-KDA CARBOXYL-TERMINAL FRAGMENT
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DOI：
10.1073/pnas.85.8.2543
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发表时间：
1988-04-01
影响因子：
11.1
通讯作者：
EARNSHAW, WC
中科院分区：
文献类型：
--
作者：
DARPA, P;MACHLIN, PS;EARNSHAW, WC
编码人拓扑异构酶I的cDNA克隆从用自身免疫抗拓扑异构酶I血清筛选的表达载体文库（gt 11）中分离。这些克隆中的一个已被表达为融合蛋白，该融合蛋白由细菌TrpE蛋白的32-kDa片段连接到由cDNA编码的67.7 kDa蛋白质组成。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)融合蛋白松弛超螺旋DNA，一种可以被抗拓扑异构酶I血清免疫沉淀的活性。(iii)序列分析表明，最长的cDNA克隆（3645个碱基对）编码765个氨基酸的蛋白质，与酿酒酵母拓扑异构酶I有42%的同源性。序列数据还表明，催化活性的67.7-kDa片段由羧基末端组成。
cDNA clones encoding human toposiomerase 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 the 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.