HUMAN DNA HELICASE-V, A NOVEL DNA UNWINDING ENZYME FROM HELA-CELLS
HUMAN DNA HELICASE-V, A NOVEL DNA UNWINDING ENZYME FROM HELA-CELLS
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DOI：
10.1093/nar/21.10.2323
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发表时间：
1993-05-25
影响因子：
14.9
通讯作者：
FALASCHI, A
中科院分区：
文献类型：
--
作者：
TUTEJA, N;RAHMAN, K;FALASCHI, A
利用P-32标记的寡核苷酸与M13单链DNA的链置换分析，我们纯化了一种新的从HeLa细胞核中解离DNA的酶，人DNA解旋酶V(HDH V)。它以极低的丰度存在于细胞中，在其他人类解旋酶中具有最高的周转率。从300g培养细胞中仅分离到0.012 mg的纯蛋白，不含DNA拓扑异构酶、连接酶、划痕和核酸酶活性。该酶还表现出依赖于单链DNA的ATPase活性，经SDS-聚丙烯酰胺凝胶电泳法测得表观分子量为92 kDa。只有三磷酸腺苷或二磷酸腺苷水解酶支持解离活性。解旋酶的活性需要二价阳离子(Mg2+和Gt；Mn2+)，其最适浓度为1.0 mM；它解开长度不到25bp的DNA双链，单链DNA的长度短至49个核苷酸。执行DNA解离不需要复制叉状结构。HDH V既不能解开钝端的双链DNA，也不能解开DNA-RNA杂交物；它通过沿结合链向3‘到5’方向移动来单向解开DNA，这一极性类似于先前描述的人类DNA解旋酶I和III(Tuteja等人)。核酸研究报告18,6785-6792,1990；Tuteja等人。核酸研究报告20,5329-5337,1992)，与人类DNA解旋酶IV相反(Tuteja等人)。核酸研究报告19,3613-3618,1991)。
Using a strand-displacement assay with P-32 labeled oligonucleotide annealed to M13 ssDNA we have purified to apparent homogeneity and characterized a novel DNA unwinding enzyme from HeLa cell nuclei, human DNA helicase V (HDH V). This is present in extremely low abundance in the cells and has the highest turnover rate among other human helicases. From 300 grams of cultured cells only 0.012 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The enzyme also shows ATPase activity dependent on single-stranded DNA and has an apparent molecular weight of 92 kDa by SDS-polyacrylamide gel electrophoresis. Only ATP or dATP hydrolysis supports the unwinding activity. The helicase requires a divalent cation (Mg2+ > Mn2+) at an optimum concentration of 1.0 mM for activity; it unwinds DNA duplexes less than 25 bp long and having a ssDNA stretch as short as 49 nucleotides. A replication fork-like structure is not required to perform DNA unwinding. HDH V cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids; it unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand, a polarity similar to the previously described human DNA helicases I and III (Tuteja et al. Nucleic Acids Res. 18, 6785 - 6792, 1990; Tuteja et al. Nucleic Acid Res. 20, 5329 - 5337, 1992) and opposite to that of human DNA helicase IV (Tuteja et al. Nucleic Acid Res. 19, 3613 - 3618, 1991).