Inhibition of lagging strand replication by G-rich telomeric DNA and the shelterin subunit POT1

Telomeres preserve stable eukaryotic chromosomes by protecting the natural chromosome ends from DNA repair but pose a persistent challenge to the replication machinery and an endogenous source of replication stress. Different features have been implicated in causing this effect but how the canonical replication process is altered at telomeres remains poorly understood. To address this question, we have reconstituted telomere replication with purified human proteins. Our system reveals that G-rich telomeric DNA can directly and specifically block lagging strand replication in a manner counteracted by BLM helicase. Unexpectedly, we also identify shelterin as inhibitory for the lagging strand. Biochemical experiments and electron microscopy imaging show that POT1-containing shelterin complexes induce Okazaki fragment skipping by binding the lagging strand template, generating large single-stranded gaps that are left behind on otherwise fully replicated molecules. Our study defines how the core components of telomeres interfere with the canonical replication process, identifying multiple potential sources of replication stress.