Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper-resection.

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context-dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single-strand DNA (ssDNA) transactions, including the ssDNA-binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1-Top3-Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.

A role for the yeast PCNA unloader Elg1 in eliciting the DNA damage checkpoint.

During cell proliferation, the genome is constantly threatened by cellular and external factors. When the DNA is damaged, or when its faithful duplication is delayed by DNA polymerase stalling, the cells induce a coordinated response termed the DNA damage response (DDR) or checkpoint. Elg1 forms an RFC-like complex in charge of unloading the DNA polymerase processively factor PCNA during DNA replication and DNA repair. Using checkpoint-inducible strains, a recently published paper (Sau et al. in mBio 10(3):e01159-19. https://doi.org/10.1128/mbio.01159-19, 2019) uncovered a role for Elg1 in eliciting the DNA damage checkpoint (DC), one of the branches of the DDR. The apical kinase Mec1/ATR phosphorylates Elg1, as well as the adaptor proteins Rad9/53BP1 and Dpb11/TopBP1, which are recruited to the site of DNA damage to amplify the checkpoint signal. In the absence of Elg1, Rad9 and Dpb11 are recruited but fail to be phosphorylated and the signal is therefore not amplified. Thus, Elg1 appears to coordinate DNA repair and the induction of the DNA damage checkpoint.

Preparation of endogenous TopBP1/Dpb11 and effect on central checkpoint kinase Mec1- Ddc2 (human ATR-ATRIP homolog).

The Saccharomyces cerevisiae Mec1 kinase, the mammalian ATR ortholog, is essential for sensing a variety of DNA lesions and initiating DNA damage response. The Dpb11, a homolog of human TopBP1, functions in activating the Mec1 upon DNA replication stress and DNA damages. Here, we report an affinity purification and ion exchange chromatography method to efficiently purify endogenous Dpb11 under normal expression level directly from yeast whole cell extraction. The final concentration of 5 µM of high purity and homogeneity biochemical preparation enables functional and structural characterization of the physical interaction between Dpb11 and Mec1-Ddc2 complex. The Dpb11 obtained by endogenous purification strongly stimulates the Mec1 kinase activity and promotes the changes in conformational distribution. This observation suggests the Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations.

The Yeast PCNA Unloader Elg1 RFC-Like Complex Plays a Role in Eliciting the DNA Damage Checkpoint.

The PCNA (proliferating cell nuclear antigen) ring plays central roles during DNA replication and repair. The yeast Elg1 RFC-like complex (RLC) is the principal unloader of chromatin-bound PCNA and thus plays a central role in maintaining genome stability. Here we identify a role for Elg1 in the unloading of PCNA during DNA damage. Using DNA damage checkpoint (DC)-inducible and replication checkpoint (RC)-inducible strains, we show that Elg1 is essential for eliciting the signal in the DC branch. In the absence of Elg1 activity, the Rad9 (53BP1) and Dpb11 (TopBP1) adaptor proteins are recruited but fail to be phosphorylated by Mec1 (ATR), resulting in a lack of checkpoint activation. The chromatin immunoprecipitation of PCNA at the Lac operator sites reveals that accumulated local PCNA influences the checkpoint activation process in elg1 mutants. Our data suggest that Elg1 participates in a mechanism that may coordinate PCNA unloading during DNA repair with DNA damage checkpoint induction.IMPORTANCE The Elg1protein forms an RFC-like complex in charge of unloading PCNA from chromatin during DNA replication and repair. Mutations in the ELG1 gene caused genomic instability in all organisms tested and cancer in mammals. Here we show that Elg1 plays a role in the induction of the DNA damage checkpoint, a cellular response to DNA damage. We show that this defect is due to a defect in the signal amplification process during induction. Thus, cells coordinate the cell's response and the PCNA unloading through the activity of Elg1.

Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling.

The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.

Slx4 scaffolding in homologous recombination and checkpoint control: lessons from yeast.

Homologous recombination-mediated DNA repair is essential for maintaining genome integrity. It is a multi-step process that involves resection of DNA ends, strand invasion, DNA synthesis and/or DNA end ligation, and finally, the processing of recombination intermediates such as Holliday junctions or other joint molecules. Over the last 15 years, it has been established that the Slx4 protein plays key roles in the processing of recombination intermediates, functioning as a scaffold to coordinate the action of structure-specific endonucleases. Recent work in budding yeast has uncovered unexpected roles for Slx4 in the initial step of DNA-end resection and in the modulation of DNA damage checkpoint signaling. Here we review these latest findings and discuss the emerging role of yeast Slx4 as an important coordinator of DNA damage signaling responses and a regulator of multiple steps in homologous recombination-mediated repair.

Multi-BRCT scaffolds use distinct strategies to support genome maintenance.

Genome maintenance requires coordinated actions of diverse DNA metabolism processes. Scaffolding proteins, such as those containing multiple BRCT domains, can influence these processes by collaborating with numerous partners. The best-studied examples of multi-BRCT scaffolds are the budding yeast Dpb11 and its homologues in other organisms, which regulate DNA replication, repair, and damage checkpoints. Recent studies have shed light on another group of multi-BRCT scaffolds, including Rtt107 in budding yeast and related proteins in other organisms. These proteins also influence several DNA metabolism pathways, though they use strategies unlike those employed by the Dpb11 family of proteins. Yet, at the same time, these 2 classes of multi-BRCT proteins can collaborate under specific situations. This review summarizes recent advances in our understanding of how these multi-BRCT proteins function in distinct manners and how they collaborate, with a focus on Dpb11 and Rtt107.

iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.

Assembly of Slx4 signaling complexes behind DNA replication forks.

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.

Behavior of replication origins in Eukaryota - spatio-temporal dynamics of licensing and firing.

Although every organism shares some common features of replication, this process varies greatly among eukaryotic species. Current data show that mathematical models of the organization of origins based on possibility theory may be applied (and remain accurate) in every model organism i.e. from yeast to humans. The major differences lie within the dynamics of origin firing and the regulation mechanisms that have evolved to meet new challenges throughout the evolution of the organism. This article elaborates on the relations between chromatin structure, organization of origins, their firing times and the impact that these features can have on genome stability, showing both differences and parallels inside the eukaryotic domain.

Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions.

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down-regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase-independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4-Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi-BRCT-domain (MBD) module that recapitulates the action of Slx4-Rtt107 in checkpoint down-regulation. MBD mimics the damage-induced Dpb11-Slx4-Rtt107 complex by synergistically interacting with lesion-specific phospho-sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11-Slx4-Rtt107 or MBD via a cooperative 'two-site-docking' mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio-temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT-domains interactions.