UC Berkeley

Linear chromosomes lose terminal sequence with every cell division due to incomplete replication by the eukaryotic replisome. Sequence must be added back to DNA ends, called telomeres, in order to deter instability that would occur as loss of end sequence encroached on genic DNA. Human telomeres are elongated by a ribonucleoprotein enzyme called telomerase, composed of the protein, TERT, and the RNA, hTR, which adds telomeric sequence onto DNA ends through reverse transcription off of its internal RNA template. Telomerase ensures indefinite replicative potential through maintenance of these ends, and is thus important for both normal cellular growth, and uncontrolled cell growth observed in most cancers.

Regulation of hTR accumulation is a multi step process involving many protein partners. Defects in the biogenesis pathway lead to reduced levels of hTR and impaired telomere maintenance. I show that the G-quadruplex resolvase DHX36 binds to hTR dependent on hTR's 5' guanosines. The binding occurs through the unique N-terminal domain of DHX36 which has demonstrated specificity for G-quadruplexes. Furthermore, stable accumulation of hTR depends on the presence of the 5' guanosines, implicating DHX36-mediated resolution of the 5' end of hTR in stable accumulation of the RNA.

Recruitment of mature telomerase to its substrate is dependent on the telomere binding protein TPP1. Specifically, it has been shown that the oligonucleotide/oligosaccharide-binding fold (OB) of TPP1 is important for recruitment of telomerase to the telomere. Using directed mutagenesis, I identified a discrete OB fold surface of TERT-TPP1 interaction, later termed the TEL patch. Importantly, the TEL patch is physically distinct from other regions that are essential for binding other shelterin proteins that maintain telomeric integrity and guard against recognition of the telomere end by DNA damage response proteins. Using genetic manipulation of human embryonic stem cells, I determined that the TEL patch is essential for recruitment to the telomere in a cellular system. Furthermore, through a separate residue, L104, TPP1 dictates the responsiveness of telomerase to telomere length. Together, these findings open up new targets for pharmaceutical inhibition of telomerase activity in cancer.