DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9

Abstract

In response to genotoxic stress, a transient arrest in cell cycle progression enforced by the DNA damage checkpoint (DDC) signaling pathway positively contributes to genome maintenance1. Because hyperactivated DDC can lead to a persistent and detrimental cell cycle arrest2,3, cells must tightly regulate the activity of DDC kinases. Despite their importance, the mechanisms for monitoring and modulating DDC signaling are not fully understood. Here we show that DNA repair scaffolding proteins Slx4 and Rtt107 prevent lesions generated during DNA replication from aberrantly hyperactivating DDC signaling in Saccharomyces cerevisiae. Upon replication stress, cells lacking Slx4 or Rtt107 exhibit hyperactivation of the downstream DDC kinase Rad53 while activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phospho-H2A, two positive regulators of Rad9-dependent Rad53 activation. Reduction of DDC signaling by hypomorphic mutations in RAD53 and H2A rescue the hyper-sensitivity of slx4Δ or rtt107Δ cells to replication stress. We propose that the Slx4-Rtt107 complex modulates Rad53 activation via a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings reveal that DDC signaling is monitored and modulated through the direct action of DNA repair factors.

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength

“…Accumulating evidence suggests that scaffold-like proteins, such as those harbouring BRCT domains 2 , are involved in the signal transduction pathways of DDRs by recruiting various components and regulators. A recent study has revealed that DNA repair scaffolds directly modulate the checkpoint signalling pathway in budding yeast 3 . Therefore, the functions of scaffoldlike proteins in the DDR play essential roles in the coordination of cellular responses to maintain homeostasis.…”

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

“…74 The latter modification, particularly at Serine 486 of Slx4, contributes to its interaction with Dpb11. 77,79 These studies also suggest that phosphorylated Slx4 likely directly binds to Dpb11, while Rtt107 stabilizes this interaction. The Slx4-Dpb11 interaction is conserved as phosphorylated human SLX4 also associates with TopBP1.…”

“…77,80,81 An underlying mechanism may be that Slx4 and Rad9 compete for binding to BRCT1-2 of Dpb11, though Slx4 can also associate with BRCT3-4 of Dpb11. 77,79 It appears that BRCT1-2 and BRCT3-4 of Dpb11 can engage Slx4 and the 9-1-1 complex. 77,79,80 As such, Dpb11 can bridge Slx4 and 9-1-1 (Fig.…”

“…77,79 It appears that BRCT1-2 and BRCT3-4 of Dpb11 can engage Slx4 and the 9-1-1 complex. 77,79,80 As such, Dpb11 can bridge Slx4 and 9-1-1 (Fig. 3).…”

“…However recent studies suggest that Rtt107, in collaboration with its partners, serves as a replication accessory protein that influences DNA repair, replisome progression and checkpoint signaling. 56,57,[75][76][77] In these capacities, these proteins are critical for genome stability, and studies of their functions have provided important insights into how replication accessory proteins facilitate genome duplication. The remaining sections describe new developments regarding Rtt107 and its partners.…”