title: Modulation of yeast telomerase activity by Cdc13 and Est1 in vitro.
abstract: Telomerase is the enzyme involved in extending telomeric DNA. Control of telomerase activity by modulating its access to chromosome ends is one of the most important fundamental mechanisms. This study established an in vitro yeast telomerase reconstitution system that resembles telomere replication in vivo. In this system, a tailed-duplex DNA formed by telomeric DNA was employed to mimic the structure of telomeres. The core catalytic components of telomerase Est2/Tlc1 RNA were used as the telomeric DNA extension machinery. Using the reconstituted systems, this study found that binding of Cdc13 to telomeric DNA inhibited the access of telomerase to its substrate. The result was further confirmed by a single-molecule approach using the tethered-particle motion (TPM)-based telomerase assay. The findings also showed that the inhibitory effect can be relieved by telomerase-associated protein Est1, consistent with the role of Cdc13 and Est1 in regulating telomere extension in vivo. Significantly, this study found that the DNA binding property of Cdc13 was altered by Est1, providing the first mechanistic evidence of Est1 regulating the access of telomerase to its substrate. Thus, the roles of Cdc13 and Est1 in modulating telomerase activity were clearly defined using the in vitro reconstituted system.
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Yu-Fan
authors.initials: YF
authors.lastname: Chen
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan.
authors.forename: Chia-Ying
authors.initials: CY
authors.lastname: Lu
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Yi-Chien
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Tai-Yuan
authors.initials: TY
authors.lastname: Yu
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan.
authors.forename: Chun-Ping
authors.initials: CP
authors.lastname: Chang
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei 100, Taiwan.
authors.forename: Jing-Ru
authors.initials: JR
authors.lastname: Li
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei 100, Taiwan.
authors.forename: Hung-Wen
authors.initials: HW
authors.lastname: Li
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan.|Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan.
authors.forename: Jing-Jer
authors.initials: JJ
authors.lastname: Lin