The Replication Checkpoint Protects Fork Stability by Releasing Transcribed Genes from Nuclear Pores

Summary

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Abstract

Graphical Abstract

Highlights

► Gene gating inactivation suppresses fork collapse in checkpoint mutants ► The Mec1 checkpoint releases transcribed chromatin from nuclear pore complexes ► Mec1 phosphorylates Mlp1 nucleoporin to release topological blocks at gated genes ► Releasing local topology by double-strand break formation counteracts fork collapse