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 in mechanistic terms remains poorly understood. To address this question, we have reconstituted telomere replication with purified human proteins. This system reveals that while G-rich telomeric DNA directly and specifically blocks lagging strand replication in a manner counteracted by BLM helicase, shelterin unexpectedly acts as an additional lagging strand barrier. 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 remain unreplicated. Our study defines how the core components of telomeres interfere with the canonical replication process, identifying several potential sources of replication stress.