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.     

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.     

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 Caenorhabditis elegans Rad17 homolog HPR-17 is required for telomere replication

Subunits of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen (PNCA)-like sliding clamp are required for DNA damage responses and telomerase-mediated telomere replication in the nematode Caenorhabditis elegans. PCNA sliding clamps are loaded onto DNA by a replication factor C (RFC) clamp loader. The C. elegans Rad17 RFC clamp loader homolog, hpr-17, functions in the same pathway as the 9-1-1 complex with regard to both the DNA damage response and telomerase-mediated telomere elongation. Thus, hpr-17 defines an RFC-like complex that facilitates telomerase activity in vivo in C. elegans.     

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1^K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9¹⁻²⁶⁶ (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9¹⁻²⁶⁶–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9¹⁻²⁶⁶–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.     

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.     

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.     

Structure-Function Analysis of Fission Yeast Hus1-Rad1-Rad9 Checkpoint Complex

Hus1, Rad1, and Rad9 are three evolutionarily conserved proteins required for checkpoint control in fission yeast. These proteins are known to form a stable complex in vivo. Recently, computational studies have predicted structural similarity between the individual proteins of Hus1-Rad1-Rad9 complex and the replication processivity factor proliferating cell nuclear antigen (PCNA). This has led to the proposal that the Hus1-Rad1-Rad9 complex may form a PCNA-like ring structure, and could function as a sliding clamp during checkpoint control. In the present study, we have attempted to test the predictions of this model by asking whether the PCNA alignment identifies functionally important residues or explains mutant phenotypes of hus1, rad1, or rad9 alleles. Although some of our results are consistent with the PCNA alignment, others indicate that the Hus1-Rad1-Rad9 complex possesses unique structural and functional features.     

Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA

The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17–Rfc2–Rfc3–Rfc4–Rfc5) complex and an RHR heterotrimer (Rad1–Hus1–Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5′ recessed ends whereas RFC preferred 3′ recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5′ recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.     

Biochemical Characterization of DNA Damage Checkpoint Complexes: Clamp Loader and Clamp Complexes with Specificity for 5′ Recessed DNA

The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17–Rfc2–Rfc3–Rfc4–Rfc5) complex and an RHR heterotrimer (Rad1–Hus1–Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5′ recessed ends whereas RFC preferred 3′ recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5′ recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.     

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.     

DNA repair by polymerase delta in Saccharomyces cerevisiae is not controlled by the proliferating cell nuclear antigen-like Rad17/Mec3/Ddc1 complex

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32Delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.     

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.     

Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185)) was identified as the only topological equivalent of PCNA(K164). To investigate a potential role of posttranslational modifications of Rad1(K185) in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R) allele. The Rad1(K185) residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185) is not a functional counterpart of PCNA(K164).     

Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy

Human checkpoint Rad proteins are thought to function as damage sensors in the DNA damage checkpoint response pathway. The checkpoint proteins hRad9, hHus1, and hRad1 have limited homology to the replication processivity factor proliferating cell nuclear antigen (PCNA), and hRad17 has homology to replication factor C (RFC). Such observations have led to the proposal that these checkpoint Rad proteins may function similarly to their replication counterparts during checkpoint control. We purified two complexes formed by the checkpoint Rad proteins and investigated their structures using an electron microscopic preparative method in which the complexes are sprayed from a glycerol solution onto very thin carbon foils, decorated in vacuo with tungsten, and imaged at low voltage. We found that the hRad9, hHus1, and hRad1 proteins make a trimeric ring structure (checkpoint 9-1-1 complex) reminiscent of the PCNA ring. Similarly we found that hRad17 makes a heteropentameric complex with the four RFC small subunits (hRad17-RFC) with a deep groove or cleft and is similar to the RFC clamp loader. Therefore, our results demonstrate structural similarity between the checkpoint Rad complexes and the PCNA and RFC replication factors and thus provide further support for models proposing analogous functions for these complexes.     

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.     

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.     

The human checkpoint Rad protein Rad17 is chromatin-associated throughout the cell cycle, localizes to DNA replication sites, and interacts with DNA polymerase epsilon

The checkpoint Rad proteins Rad17, Rad9, Rad1, Hus1, ATR, and ATRIP become associated with chromatin in response to DNA damage caused by genotoxic agents and replication inhibitors, as well as during unperturbed DNA replication in S phase. Here we show that murine Rad17 is phosphorylated at two sites that were previously shown to be modified in response to DNA damage, independent of DNA damage and ATM, in proliferating tissue. In contrast to studies with Xenopus laevis extracts but similar to observations in Schizosaccharomyces pombe, the level of chromatin-bound hRad17 remains relatively constant during the cell cycle and does not change significantly in response to DNA damage or replication block. However, phosphorylated hRad17 preferentially associates with the sites of ongoing DNA replication and interacts with the DNA replication protein, DNA polymerase ε. These results provide a link between the DNA damage checkpoint machinery and the replication apparatus and suggest that hRad17 may play a role in monitoring the progress of DNA replication via its interaction with DNA polymerase ε.     

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.