Characterization of Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1 and Rad9

Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9(+) locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.     

The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage.

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutSα and hMutLα. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.     

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.     

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.     

Crystal Structure of the Human Rad9-Hus1-Rad1 Clamp

Three evolutionarily conserved proteins, Rad9, Hus1, and Rad1, form a heterotrimeric 9-1-1 complex that plays critical roles in cellular responses to DNA damage by activating checkpoints and by recruiting DNA repair enzymes to DNA lesions. We have determined the crystal structure of the human Rad9 (residues 1-272)-Hus1-Rad1 complex at 2.5 Å resolution. The 9^1-272-1-1 complex forms a closed ring, with each subunit having a similar structure. Despite its high level of similarity to proliferating cell nucleus antigen in terms of overall structure, the 9^1-272-1-1 complex exhibits notable differences in local structures, including interdomain connecting loops, H2 and H3 helices, and loops in the vicinity of the helices of each subunit. These local structural variations provide several unique features to the 9-1-1 heterotrimeric complex—including structures of intermolecular interfaces and the inner surface around the central hole, and different electrostatic potentials at and near the interdomain connecting loops of each 9-1-1 subunit—compared to the proliferating cell nucleus antigen trimer. We propose that these structural features allow the 9-1-1 complex to bind to a damaged DNA during checkpoint control and to serve as a platform for base excision repair. We also show that the 9^1-272-1-1 complex, but not the full-length 9-1-1 complex, forms a stable complex with the 5′ recessed DNA, suggesting that the C-terminal tail of Rad9 is involved in the regulation of the 9-1-1 complex in DNA binding.     

A Ddc2-Rad53 fusion protein can bypass the requirements for RAD9 and MRC1 in Rad53 activation

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a “mediator” protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Δrad9 or Δrad9Δmrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Δrad9Δmrc1 cells. In addition, expression of Ddc2-Rad53p in Δrad24Δrad17Δmec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.     

Opening pathways of the DNA clamps proliferating cell nuclear antigen and Rad9-Rad1-Hus1

Proliferating cell nuclear antigen and the checkpoint clamp Rad9-Rad1-Hus1 topologically encircle DNA and act as mobile platforms in the recruitment of proteins involved in DNA damage response and cell cycle regulation. To fulfill these vital cellular functions, both clamps need to be opened and loaded onto DNA by a clamp loader complex—a process, which involves disruption of the DNA clamp’s subunit interfaces. Herein, we compare the relative stabilities of the interfaces using the molecular mechanics Poisson−Boltzmann solvent accessible surface method. We identify the Rad9-Rad1 interface as the weakest and, therefore, most likely to open during clamp loading. We also delineate the dominant interface disruption pathways under external forces in multiple-trajectory steered molecular dynamics runs. We show that, similar to the case of protein folding, clamp opening may not proceed through a single interface breakdown mechanism. Instead, we identify an ensemble of opening pathways, some more prevalent than others, characterized by specific groups of contacts that differentially stabilize the regions of the interface and determine the spatial and temporal patterns of breakdown. In Rad9-Rad1-Hus1, the Rad9-Rad1 and Rad9-Hus1 interfaces share the same dominant unzipping pathway, whereas the Hus1-Rad1 interface is disrupted concertedly with no preferred directionality.     

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.     

ATR,Claspin and the Rad9-Rad1-Hus1 Complex Regulate Chk1 and Cdc25A in the Absence of DNA Damage

The ATR and Chk1 kinases are essential to maintain genomicintegrity. ATR, with Claspin and the Rad9-Rad1-Hus1 complex,activates Chk1 after DNA damage. Chk1-mediated phosphorylation ofthe Cdc25A phosphatase is required for the mammalian S-phasecheckpoint. Here, we show that during physiological S phase theregulation of the Chk1-Cdc25A pathway depends on ATR, Claspin,Rad9, and Hus1. Human cells with chemically or genetically ablatedATR showed inhibition of Chk1-dependent phosphorylation of Cdc25A,and they accumulated Cdc25A without external DNA damage.Furthermore, siRNA-mediated depletion of Claspin, Rad9 and Hus1also stabilized Cdc25A. ATR ablation also inhibited the activatoryphosphorylation of Chk1 on serine 345. Thus, the ATR-Chk1-Cdc25Apathway represents an integral part of physiological S-phaseprogression, and interference with this mechanism underminesviability of somatic mammalian cells. DNA damage further activatesand switches this pathway from its constitutively operating“surveillance mode” compatible with DNA replication into an“emergency” checkpoint response.     

Mutants defective in Rad1-Rad10-Slx4 exhibit a unique pattern of viability during mating-type switching in Saccharomyces cerevisiae

Efficient repair of DNA double-strand breaks (DSBs) requires the coordination of checkpoint signaling and enzymatic repair functions. To study these processes during gene conversion at a single chromosomal break, we monitored mating-type switching in Saccharomyces cerevisiae strains defective in the Rad1-Rad10-Slx4 complex. Rad1-Rad10 is a structure-specific endonuclease that removes 3' nonhomologous single-stranded ends that are generated during many recombination events. Slx4 is a known target of the DNA damage response that forms a complex with Rad1-Rad10 and is critical for 3'-end processing during repair of DSBs by single-strand annealing. We found that mutants lacking an intact Rad1-Rad10-Slx4 complex displayed RAD9- and MAD2-dependent cell cycle delays and decreased viability during mating-type switching. In particular, these mutants exhibited a unique pattern of dead and switched daughter cells arising from the same DSB-containing cell. Furthermore, we observed that mutations in post-replicative lesion bypass factors (mms2Delta, mph1Delta) resulted in decreased viability during mating-type switching and conferred shorter cell cycle delays in rad1Delta mutants. We conclude that Rad1-Rad10-Slx4 promotes efficient repair during gene conversion events involving a single 3' nonhomologous tail and propose that the rad1Delta and slx4Delta mutant phenotypes result from inefficient repair of a lesion at the MAT locus that is bypassed by replication-mediated repair.     

Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex

The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.     

The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase

The checkpoint protein Rad9/Rad1/Hus1 heterotrimer (the 9-1-1 complex) is structurally similar to the proliferating cell nuclear antigen sliding clamp and has been proposed to sense DNA damage that leads to cell cycle arrest or apoptosis. Human (h) NEIL1 DNA glycosylase, an ortholog of bacterial Nei/Fpg, is involved in repairing oxidatively damaged DNA bases. In this study, we show that hNEIL1 interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. Residues 290–350 of hNEIL1 are important for the 9-1-1 association. A significant fraction of the hNEIL1 nuclear foci co-localize with hRad9 foci in hydrogen peroxide treated cells. Human NEIL1 DNA glycosylase activity is significantly stimulated by hHus1, hRad1, hRad9 separately and the 9-1-1 complex. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of base excision repair.     

Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Delta mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.     

Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast

In mammalian and budding yeast cells treated with genotoxic agents, different proteins implicated in detecting, signalling or repairing DNA lesions form nuclear foci. We studied foci formed by proteins involved in these processes in living fission yeast cells, which is amenable to genetic and molecular analysis. Using fluorescent tags, we analysed subnuclear localisations of the DNA damage checkpoint protein Rad9, of the homologous recombination protein Rad22 and of PCNA, which are implicated in many aspects of DNA metabolism. After inducing double strand breaks (DSBs) with ionising radiations, Rad22, Rad9 and PCNA form a low number of nuclear foci. Rad9 recruitment to foci depends on the presence of Rad1, Hus1 and Rad17, but is independent of downstream checkpoint effectors and of homologous recombination proteins. Likewise, Rad22 and PCNA form foci despite inactive homologous recombination repair and impaired DNA damage checkpoint. Rad22 and Rad9 foci co-localise completely, whereas PCNA co-localises with Rad22 and Rad9 only partially. Foci do not disassemble in cells unable to repair DNA by homologous recombination. Thus, in fission yeast, DSBs are detected by the DNA damage checkpoint and are repaired by homologous recombination at a few spatially confined subnuclear compartments where Rad22, Rad9 and PCNA concentrate independently.     

A role of the C-terminal region of human Rad9 (hRad9) in nuclear transport of the hRad9 checkpoint complex

Rad9, Rad1, and Hus1 are members of the Rad family of checkpoint proteins that are required for both DNA replication and DNA damage checkpoints and are thought to function as sensors in the DNA integrity checkpoint control. These proteins can interact with each other and form a stable proliferating cell nuclear antigen-related Rad9.Rad1.Hus1 heterotrimeric complex that might encircle DNA at or near the damaged sites. In this study, we demonstrate that the human Rad9 (hRad9) protein contains a predicted nuclear localization sequence (NLS) near its C terminus, which plays an essential role in the hRad9-mediated G(2) checkpoint. Deletion experiments indicate that the NLS-containing region of hRad9 is critical for the nuclear transport of not only hRad9 but also human Rad1 (hRad1) and human Hus1 (hHus1), although this region is not required for hRad9.hRad1.hHus1 complex formation. In support of the role that hRad9 NLS plays in the nuclear targeting of the hRad9.hRad1.hHus1 complex, overexpression of a deletion mutant of hRad9 lacking the NLS-containing C-terminal region can bypass the G(2) checkpoint and result in cell death after ionizing radiation or hydroxyurea treatment. Moreover, knockdown of hRad9 expression by small interfering RNA (siRNA) results in hRad1 accumulation in the cytoplasm and significantly abrogates the G(2) checkpoint in the presence of damaged DNA or incomplete DNA replication. Thus, the C-terminal region of human Rad9 protein is important for G(2) checkpoint control by operating the transport of the hRad9.hRad1.hHus1 checkpoint complex into the nucleus.     

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.