title: The C-terminal extension of human telomerase reverse transcriptase is necessary for high affinity binding to telomeric DNA.
abstract: The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from short telomere syndromes, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic analyses and molecular modelling of a disease-associated mutant in the C-terminal extension of the reverse transcriptase subunit of human telomerase. The kinetic analyses revealed that the mutation substantially impacts the affinity of telomerase for telomeric DNA, but the magnitude of this impact varies for primers with different 3' ends. Molecular dynamics simulations corroborate this finding, revealing that the mutation results in greater movement of a nearby loop, impacting the DNA-RNA helix differentially with different DNA primers. Thus, the data indicate that this region is the location of one of the enzyme conformational changes responsible for the long-standing observation that off-rates of telomerase vary with telomeric 3' end sequence. Our data provide a molecular basis for a disease-associated telomerase mutation, and the first direct evidence for a role of the C-terminal extension in DNA binding affinity, a function analogous to the "thumb" domain of retroviral reverse transcriptases.
authors.affiliation: Children's Medical Research Institute, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Tomlinson
authors.affiliation: ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia.
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Holien
authors.affiliation: Children's Medical Research Institute, University of Sydney, Westmead, NSW, 2145, Australia.
authors.forename: Jordan A T
authors.initials: JA
authors.lastname: Mathias
authors.affiliation: ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, 3010, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: Children's Medical Research Institute, University of Sydney, Westmead, NSW, 2145, Australia. Electronic address: tbryan@cmri.org.au.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Bryan
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D019098:Telomerase / Q000737:chemistry* / Q000378:metabolism; D016615:Telomere / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D004247:DNA; C509186:TERT protein, human; D019098:Telomerase
keywords: Biochemistry; Cancer; Molecular modelling; Short telomere syndromes; Telomerase