Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9^ΔC-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9^ΔC-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.     

Rad9, Rad17, TopBP1 and Claspin Play Essential Roles in Heat-Induced Activation of ATR Kinase and Heat Tolerance

Hyperthermia is widely used to treat patients with cancer, especially in combination with other treatments such as radiation therapy. Heat treatment per se activates DNA damage responses mediated by the ATR-Chk1 and ATM-Chk2 pathways but it is not fully understood how these DNA damage responses are activated and affect heat tolerance. By performing a genetic analysis of human HeLa cells and chicken B lymphoma DT40 cells, we found that heat-induced Chk1 Ser345 phosphorylation by ATR was largely dependent on Rad9, Rad17, TopBP1 and Claspin. Activation of the ATR-Chk1 pathway by heat, however, was not associated with FancD2 monoubiquitination or RPA32 phosphorylation, which are known as downstream events of ATR kinase activation when replication forks are stalled. Downregulation of ATR, Rad9, Rad17, TopBP1 or Claspin drastically reduced clonogenic cell viability upon hyperthermia, while gene knockout or inhibition of ATM kinase reduced clonogenic viability only modestly. Suppression of the ATR-Chk1 pathway activation enhanced heat-induced phosphorylation of Chk2 Thr68 and simultaneous inhibition of ATR and ATM kinases rendered severe heat cytotoxicity. These data indicate that essential factors for activation of the ATR-Chk1 pathway at stalled replication forks are also required for heat-induced activation of ATR kinase, which predominantly contributes to heat tolerance in a non-overlapping manner with ATM kinase.     

Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.     

The Rad4(TopBP1) ATR-activation domain functions in G1/S phase in a chromatin-dependent manner

DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4(TopBP1) AAD-defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage-independent assay for checkpoint activation that is Rad4(TopBP1) AAD-dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb2(53BP1). Consistent with a model where Rad4(TopBP1) AAD-dependent checkpoint activation is ssDNA/RPA-independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4(TopBP1) AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4(TopBP1) AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD-dependent Rad3(ATR) checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein-chromatin interactions.     

Interaction between Rad9–Hus1–Rad1 and TopBP1 activates ATR–ATRIP and promotes TopBP1 recruitment to sites of UV-damage

The checkpoint clamp Rad9–Hus1–Rad1 (9–1–1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9–1–1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9–1–1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.     

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.     

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.     

Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex

Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.     

Interactions of Human Mismatch Repair Proteins MutS�� and MutL�� with Proteins of the ATR-Chk1 Pathway

At clinically relevant doses, chemotherapeutic S_N1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSα and MutLα with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.     

Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event

Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small replication factor C (RFC) subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9-inducible phosphorylation site (Ser-272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent posttranslational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.     

The Rad9A checkpoint protein is required for nuclear localization of the claspin adaptor protein

The interaction between the 911 complex, via Rad9A, and Claspin is required for activation of the Chk1-mediated checkpoint response, along with ATR, TopBP1, and the 911 clamp loader complex Rad17/RFC. Despite the importance of the Rad9A-Claspin interaction in the cell cycle, this interaction has yet to be characterized. In this work we show this interaction persists in a variety of different conditions. During the course of this study we also determined the nuclear localization of Rad9A affected the localization of the Claspin protein, leading us to the conclusion that Rad9A is able to affect Claspin cellular localization. This was verified experimentally using a Rad9A-null cell line and reconstitution of WT Rad9A. We also show that in mES cells the Rad9A paralog, Rad9B, is also capable of affecting Claspin localization. Together, these data suggest that Rad9 plays a role in locating Claspin to sites of DNA damage, facilitating its role during the Chk1-mediated checkpoint response. Since disruption of both Rad9A and Claspin has been shown to abolish Chk1 activation, we postulate that Rad9A-mediated Claspin localization is a vital step during checkpoint activation.     

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.Key words: Rif1, TopBP1, ATR, Chk1, cell cycle control, checkpoint mechanisms, Xenopus egg extract     

Chk1 Activation Protects Rad9A from Degradation as Part of a Positive Feedback Loop during Checkpoint Signalling

Phosphorylation of Rad9A at S387 is critical for establishing a physical interaction with TopBP1, and to downstream activation of Chk1 for checkpoint activation. We have previously demonstrated a phosphorylation of Rad9A that occurs at late time points in cells exposed to genotoxic agents, which is eliminated by either Rad9A overexpression, or conversion of S387 to a non-phosphorylatable analogue. Based on this, we hypothesized that this late Rad9A phosphorylation is part of a feedback loop regulating the checkpoint. Here, we show that Rad9A is hyperphosphorylated and accumulates in cells exposed to bleomycin. Following the removal of bleomycin, Rad9A is polyubiquitinated, and Rad9A protein levels drop, indicating an active degradation process for Rad9A. Chk1 inhibition by UCN-01 or siRNA reduces Rad9A levels in cells synchronized in S-phase or exposed to DNA damage, indicating that Chk1 activation is required for Rad9A stabilization in S-phase and during checkpoint activation. Together, these results demonstrate a positive feedback loop involving Rad9A-dependend activation of Chk1, coupled with Chk1-dependent stabilization of Rad9A that is critical for checkpoint regulation.     

The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR

TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR.     

Mcm10 interacts with Rad4/Cut5(TopBP1) and its association with origins of DNA replication is dependent on Rad4/Cut5(TopBP1)

Initiation of DNA replication in eukaryotes is a highly conserved and ordered process involving the co-ordinated, stepwise association of distinct proteins at multiple origins of replication throughout the genome. Here, taking Schizosaccharomyces pombe as a model, the role of Rad4(TopBP1) in the assembly of the replication complex has been examined. Quantitative chromatin immunoprecipitation experiments confirm that Rad4(TopBP1) associates with origins of DNA replication and, in addition, demonstrate that the protein is not present within the active replisome. A direct interaction between Rad4(TopBP1) and Mcm10 is shown and this is reflected in the Rad4(TopBP1)-dependent origin association of Mcm10. Rad4(TopBP1) is also shown to interact with Sld2 and Sld3 and to be required for the stable origin association of these two proteins. Rad4(TopBP1) chromatin association at stalled replication forks was found to be dependent upon the checkpoint protein Rad9, which was not required for Rad4(TopBP1) origin association. Comparison of the levels of chromatin association at origins of replication and stalled replication forks and the differential requirement for Rad9 suggest functional differences for Rad4(TopBP1) at these distinct sites.     

Claspin,a Chk1-regulatory protein,monitors DNA replication on chromatin independently of RPA,ATR, and Rad17

Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. We show here that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and Rad17 require RPA for association with DNA. Claspin, ATR, and Rad17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork.     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.     

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.     

Ubiquitin-specific Peptidase 20 Regulates Rad17 Stability,Checkpoint Kinase 1 Phosphorylation and DNA Repair by Homologous Recombination

Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.     

Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication

Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.