Oral Presentation 40th Annual Lorne Genome Conference 2019

Chromosome structure at telomeres: resolution of G-quadruplexes by telomerase (#25)

Tracy M Bryan 1 , Bishnu Paudel 2 , Aaron L Moye 1 , Hala Abou Assi 3 , Scott B Cohen 1 , Carlos Gonzalez 4 , Jennifer L Beck 2 , Masad Damha 3 , Antoine M van Oijen 2
  1. Children's Medical Research Institute, Westmead, NSW, Australia
  2. University of Wollongong, Wollongong, NSW, Australia
  3. McGill University, Montreal, Canada
  4. Instituto de Quımica Fısica ‘Rocasolano’, CSIC, Madrid, Spain

G-quadruplexes are four-stranded nucleic acid structures found at G-rich regions of the genome, including promoters, origins of replication and telomeres. It is becoming increasingly recognised that G-quadruplexes play specific roles in the regulation of biological processes such as transcription, translation and DNA replication1. Formation of G-quadruplexes is dynamic and regulated by proteins that assist DNA folding or mediate G-quadruplex resolution. Telomeric G-quadruplexes were long believed to form a protective “cap” at telomeres, preventing their extension by the ribonucleoprotein enzyme telomerase. This has led to the development of G-quadruplex stabilising ligands as potential telomerase inhibitors. Contrary to this belief, we have demonstrated that distinct subsets of telomeric G-quadruplexes can be extended by human and ciliate telomerase enzymes2,3. However, a mechanistic understanding of how telomerase deals with structured DNA has remained elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) microscopy and telomerase enzymology we provide direct evidence, at single-molecule resolution, that human telomerase can resolve and then extend parallel G-quadruplexes, and we provide a mechanism for this unexpected activity of telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex, which maintains a mostly folded structure. With the G-quadruplex correctly aligned, nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation that the G-quadruplex structure is completely unfolded to a linear product. In contrast to prevailing hypotheses, parallel G-quadruplex stabilisation with small molecule ligands did not inhibit G-quadruplex extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase. The conserved ability of human and ciliate telomerase enzymes to unwind these structures implies that parallel G-quadruplexes form at telomeres in vivo, and do not form a barrier to telomerase extension.

  1. Rhodes, D. & Lipps, H. J. G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res. 43, 8627-8637, doi:10.1093/nar/gkv862 (2015).
  2. Oganesian, L., Moon, I. K., Bryan, T. M. & Jarstfer, M. B. Extension of G-quadruplex DNA by ciliate telomerase. EMBO J. 25, 1148-1159 (2006).
  3. Moye, A. L., Porter, K. C., Cohen, S. B., Phan, T., Zyner, K. G., Sasaki, N., Lovrecz, G. O., Beck, J. L. & Bryan, T. M. Telomeric G-quadruplexes are a substrate and site of localization for human telomerase. Nat. Commun 6, 7643 (2015).