Takai, H.; Aria, V.; Borges, P.; Yeeles, J. T. P.; de Lange, T.

Telomerase solves the end-replication problem by adding G-rich telomeric repeats to the 3\' ends of telomeres1. We report a second end-replication problem affecting the C-rich telomeric repeat strand that is solved by fill-in synthesis by CST--Polymerase(Pol)-primase. In vitro replication of linear DNAs showed that lagging-strand DNA synthesis stops >40 nt before the replisome reaches the 5\' end of the template. Incomplete lagging-strand DNA synthesis is predicted to result in progressive shortening of the telomeric C-strand. Two assays to measure C-strand shortening at telomere ends in telomerase-deficient cells lacking the Ctc1 subunit of CST--Pol-primase revealed that lagging-end telomeres lose C-strand sequences at ~60 nt/population doubling (PD), consistent with the in vitro replication data. The C-strands of leading-end telomeres shortened by ~108 nt/PD, consistent with resection converting the blunt ends to 3\' overhangs. The overall shortening rate of the C-rich telomeric strand in Ctc1-deficient cells was consistent with the combined effects of incomplete lagging-strand synthesis and 5\' end resection at the leading-ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-rich strand and CST--Pol-primase to maintain the C-rich strand.