The Nuclear Pore Complex as a molecular hub to regulate replication fork repair pathways – NIRO

Replication stress impacts fork progression and leads to chromosomal instability with profound impact on development, tissue homeostasis, neurological functions and aging. Although most signalling and repair pathways are now well described, the temporal and spatial regulation of distinct fork-repair pathways remains largely elusive. Resolving replication stress occurs within a compartmentalized nucleus, with a critical role of nuclear pores complexes (NPCs) in repairing hard-to-repair DNA lesions.

This consortium has robust data indicating that replication forks stalled by specific Replication Fork Barriers (RFB) or telomere repeats relocate to NPC to favour error-free processing and fork-restart. It remains however far from clear how stressed forks are detected to relocate to NPC and how NPC contributes to the dynamic resolution of stressed forks. To fill this gap, NIRO will establish a cross-disciplinary approach that combines novel and cutting-edge functional genomics with powerful genetics assays, and live-cell imaging. The converging data of the consortium indicate that NPC allows to engage mechanisms to maintain fork integrity and replication competence.

The proposal will address how NPC mechanistically operates at stressed forks and reveal how it is linked to distinct fork-repair pathways using models of fork impediments and at the genome-wide level. The NIRO proposal is built up around two main axes.

WP1: Signaling pathways of relocation and anchoring to NPC.

The type of stressed forks that relocate to NPC and the identity of the anchoring factors are unknown. Preliminary data indicate that the dynamics relocation of stalled forks to NPC differs from those of DSBs and eroded telomeres. The consortium will develop novel and alternative methods to address transient and dynamics interactions between NPC and stressed forks both at site-specific obstacles and at the genome-wide level, and then interrogate the factors necessary to relocate and anchor stalled forks to NPC.

WP2: Mechanisms activated at NPC to preserve fork-integrity, replication competence and telomere maintenance.

NPC are instrumental to allow alternative DNA repair pathway to occur by mechanisms that remain unknown. NIRO will decipher the precise molecular transactions occurring at NPC to regulate distinct fork-repair pathways during physiological processes such as replicative senescence and recovery from replication stress.

One strength of NIRO is to address the role of NPC in resolving forks challenged by distinct obstacles and at distinct locations in the genome to obtain a broad understanding of the impact of the nuclear architecture on fork-repair pathways. We anticipate that the type of fork-obstacles and genomic location influence the signals triggering relocation and the mechanisms by which NPC acts at stressed forks. We will extend our analysis to the genome-wide level by addressing the genomic binding sites of NPC components in response to replication-blocking agents.

Given that the overall objective of NIRO is to investigate how NPC acts as a molecular hub to regulate distinct fork repair pathways, the NIRO consortium brings together three teams and their know-how in mechanisms of homologous recombination, telomere maintenance and DNA replication. One strength of this consortium is to bring highly complementary expertise’s to reveal at an unprecedented level of resolution the role of NPC in repairing replicative damage. Individual nucleoporins have been linked to human diseases but there is no general concept linking the lack of NPC functions to pathological situations. Thus, our project will open new avenues of exploration on the role of NPC in human metabolism and diseases.