期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 38, 页码 E5544-E5551出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1605517113
关键词
Tudor domain-containing domain 3; DNA topoisomerases; RNA topoisomerases; RNA circles; RNA duplex
资金
- Ministry of Science and Technology Taiwan [MOST-104-2321-B-001-012]
- Academia Sinica intramural funding [AS-105-TP-B04]
Topoisomerase 3 beta (Top3 beta) can associate with the mediator protein Tudor domain-containing protein 3 (TDRD3) to participate in two gene expression processes of transcription and translation. Despite the apparent importance of TDRD3 in binding with Top3 beta and directing it to cellular compartments critical for gene expression, the biochemical mechanism of how TDRD3 can affect the functions of Top3 beta is not known. We report here sensitive biochemical assays for the activities of Top3 beta on DNA and RNA substrates in resolving topological entanglements and for the analysis of TDRD3 functions. TDRD3 stimulates the relaxation activity of Top3 beta on hypernegatively supercoiled DNA and changes the reaction from a distributive to a processive mode. Both supercoil retention assays and binding measurement by fluorescence anisotropy reveal a heretofore unknown preference for binding single-stranded nucleic acids over duplex. Whereas TDRD3 has a structure-specific binding preference, it does not discriminate between DNA and RNA. This unique property for binding with nucleic acids can have an important function in serving as a hub to form nucleoprotein complexes on DNA and RNA. To gain insight into the roles of Top3 beta on RNA metabolism, we designed an assay by annealing two single-stranded RNA circles with complementary sequences. Top3 beta is capable of converting two such single-stranded RNA circles into a double-stranded RNA circle, and this strand-annealing activity is enhanced by TDRD3. These results demonstrate that TDRD3 can enhance the biochemical activities of Top3 beta on both DNA and RNA substrates, in addition to its function of targeting Top3 beta to critical sites in subcellular compartments.
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