The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity

DNA damage leads to activation of several mechanisms such as DNA repair and cell-cycle checkpoints. It is evident that these different cellular mechanisms have to be finely co-ordinated. Growing evidence suggests that the Rad9/Rad1/Hus1 cell-cycle checkpoint complex (9-1-1 complex), which is recruited to DNA lesion upon DNA damage, plays a major role in DNA repair. This complex has been shown to interact with and stimulate several proteins involved in long-patch base excision repair. On the other hand, the well-characterised DNA clamp-proliferating cell nuclear antigen (PCNA) also interacts with and stimulates several of these factors. In this work, we compared the effects of the 9-1-1 complex and PCNA on flap endonuclease 1 (Fen1). Our data suggest that PCNA and the 9-1-1 complex can independently bind to and activate Fen1. Finally, acetylation of Fen1 by p300-HAT abolished the stimulatory effect of the 9-1-1 complex but not that of PCNA, suggesting a possible mechanism of regulation of this important repair pathway.     

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.     

高质量抗体揭示Rad1蛋白在细胞中新的潜在活动机理

一组在进化上(从酵母到人)保守的基因Rad9、Rad1和Hus1在细胞周期监控点调控和DNA损伤修复中发挥重要作用．这三个蛋白可以形成环形异源三聚体,即9-1-1蛋白复合体．9-1-1复合体被认为是Rad9、Rad1和Hus1行使功能的主要形式．到目前为止,没有一个好的抗Rad1的抗体,严重阻碍了对Rad1和9-1-1复合体的研究．在本研究中,我们成功地制备了一株小鼠抗Rad1蛋白的单克隆抗体．这个抗体能够有效地检测小鼠和人的内源Rad1蛋白,可以用于酶联免疫吸附、蛋白质免疫印迹、免疫共沉淀和免疫荧光等实验．利用该抗体,我们发现在DNA损伤剂羟基脲(HU)的诱导下,小鼠Rad1蛋白在Rad9^+/+小鼠胚胎干细胞中表达明显增加,而在Rad9^-/-的小鼠胚胎干细胞中没有观察到该现象,这表明Rad9对Rad1的蛋白表达有调控作用．此外,内源的Rad1蛋白主要分布在细胞质中,在HU处理后并没有迁移进入细胞核的现象,这与先前广泛被人们所接受的在DNA损伤压力下Rad1和Hus1能够迁移进入细胞核并与Rad9形成9-1-1蛋白复合体的说法相矛盾．综合看来,Rad1和9-1-1蛋白复合体的分子作用机制比预期的要复杂,我们成功制备的Rad1单克隆抗体将成为研究Rad1以及9-1-1蛋白复合体的强有力的工具．     

Srs2 enables checkpoint recovery by promoting disassembly of DNA damage foci from chromatin

Following DNA repair, checkpoint signalling must be abated to resume cell cycling in a phenomenon known as checkpoint recovery. Although a number of genes have been implicated in the recovery process, it is still unknown whether checkpoint recovery is caused by a signalling network activated by DNA repair or whether it is the result of the loss of DNA structures that elicit the checkpoint. Here we show that checkpoint recovery can be uncoupled from bulk chromosome DNA repair if single-stranded (ss) DNA persists. This situation occurs in cells that are deficient in the Srs2 helicase, a protein that antagonizes Rad51. We report that srs2Δ cells fail to eliminate Ddc2 and RPA subnuclear foci following bulk chromosome repair due to the persistence of ssDNA. In contrast to cells with DNA double-strand breaks that remain unrepaired, srs2Δ cells remove the 9-1-1 checkpoint clamp from chromatin after repair. However, despite the loss of the 9-1-1 clamp, Dpb11 remains associated with chromatin to promote checkpoint activity. Our work indicates that Srs2 promotes checkpoint recovery by removing Rad51 after DNA repair. A failure to remove Rad51 causes persistence of ssDNA and the checkpoint signal. Therefore, we conclude that cells initiate recovery when the DNA structures that elicit the checkpoint are eliminated.     

ATM-dependent phosphorylation of the checkpoint clamp regulates repair pathways and maintains genomic stability

Upon genotoxic stress and during normal S phase, ATM phosphorylates the checkpoint clamp protein Rad9 in a manner that depends on Ser272. Ser272 is the only known ATM-dependent phosphorylation site in human Rad9. However, Ser272 phosphorylation is not required for survival or checkpoint activation after DNA damage. The physiological function of Ser272 remains elusive. Here, we show that ATM-dependent Rad9^Ser272 phosphorylation requires the MRN complex and controls repair pathways. Furthermore, the mutant cells accumulate large numbers of chromosome breaks and induce gross chromosomal rearrangements. Our findings establish a new and unexpected role for ATM: it phosphorylates the checkpoint clamp in order to control repair pathways, thereby maintaining genomic integrity during unperturbed cell cycle and upon DNA damage.     

ATM-dependent phosphorylation of the checkpoint clamp regulates repair pathways and maintains genomic stability

Upon genotoxic stress and during normal S phase, ATM phosphorylates the checkpoint clamp protein Rad9 in a manner that depends on Ser272. Ser272 is the only known ATM-dependent phosphorylation site in human Rad9. However, Ser272 phosphorylation is not required for survival or checkpoint activation after DNA damage. The physiological function of Ser272 remains elusive. Here, we show that ATM-dependent Rad9^Ser272 phosphorylation requires the MRN complex and controls repair pathways. Furthermore, the mutant cells accumulate large numbers of chromosome breaks and induce gross chromosomal rearrangements. Our findings establish a new and unexpected role for ATM: it phosphorylates the checkpoint clamp in order to control repair pathways, thereby maintaining genomic integrity during unperturbed cell cycle and upon DNA damage.     

Nuclear translocation promotes proteasomal degradation of human Rad17 protein through the N-terminal destruction boxes

The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230–270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.     

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.     

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.     

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.     

细胞周期监控点蛋白Rad9与非同源末端连接修复蛋白Ku70的相互作用研究

Rad9是一种重要的细胞周期监控点调控蛋白．越来越多的证据显示,Rad9也可与多种DNA损伤修复通路中的蛋白质相互作用,并调节其功能,在DNA损伤修复中发挥重要作用．非同源末端连接修复是DNA双链断裂的一条重要修复途径．Ku70、Ku80和DNA依赖的蛋白激酶催化亚基(DNA-PKcs)共同组成DNA依赖的蛋白激酶复合物(DNA-PK),在非同源末端修复连接中起重要作用．本研究中,检测到Rad9与Ku70有直接的物理相互作用和功能相互作用．我们在不同的细胞模型中发现,Rad9基因敲除、Rad9蛋白去除或Rad9表达降低会导致非同源末端连接效率明显下降．已有的研究表明,DNA损伤可导致细胞中Ku70与染色质结合增加及DNA-PKcs激酶活性增强．我们的结果显示,与野生小鼠细胞相比,Rad9基因敲除的小鼠细胞中, DNA损伤诱导的上述效应均减弱．综上所述,我们的研究首次报道了Rad9与非同源末端连接修复蛋白Ku70间有相互作用,并提示Rad9可通过调节Ku70/Ku80/DNA-PKcs复合物功能参与非同源末端连接修复．     

A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.     

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.     

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.     

Checkpoint-dependent phosphorylation of Exo1 modulates the DNA damage response

Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. In budding yeast and mice, Exo1 creates single-stranded DNA (ssDNA) at uncapped telomeres. This ssDNA accumulation activates the checkpoint response resulting in cell cycle arrest. Here, we demonstrate that Exo1 is phosphorylated when telomeres are uncapped in cdc13-1 and yku70Delta yeast cells, and in response to the induction of DNA damage. After telomere uncapping, Exo1 phosphorylation depends on components of the checkpoint machinery such as Rad24, Rad17, Rad9, Rad53 and Mec1, but is largely independent of Chk1, Tel1 and Dun1. Serines S372, S567, S587 and S692 of Exo1 were identified as targets for phosphorylation. Furthermore, mutation of these Exo1 residues altered the DNA damage response to uncapped telomeres and camptothecin treatment, in a manner that suggests Exo1 phosphorylation inhibits its activity. We propose that Rad53-dependent Exo1 phosphorylation is involved in a negative feedback loop to limit ssDNA accumulation and DNA damage checkpoint activation.     

The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency: implications for DNA repair

In eukaryotic cells, checkpoints are activated in response to DNA damage. This requires the action of DNA damage sensors such as the Rad family proteins. The three human proteins Rad9, Rad1 and Hus1 form a heterotrimeric complex (called the 9-1-1 complex) that is recruited onto DNA upon damage. DNA damage also triggers the recruitment of DNA repair proteins at the lesion, including specialized DNA polymerases. In this work, we showed that the 9-1-1 complex can physically interact with DNA polymerase β in vitro. Functional analysis revealed that the 9-1-1 complex had a stimulatory effect on DNA polymerase β activity. However, the presence of 9-1-1 complex neither affected DNA polymerase λ, another X family DNA polymerase, nor the two replicative DNA polymerases α and δ. DNA polymerase β stimulation resulted from an increase in its affinity for the primer–template and the interaction with the 9-1-1 complex stimulated deoxyribonucleotides misincorporation by DNA polymerase β. In addition, the 9-1-1 complex enhanced DNA strand displacement synthesis by DNA polymerase β on a 1 nt gap DNA substrate. Our data raise the possibility that the 9-1-1 complex might attract DNA polymerase β to DNA damage sites, thus connecting directly checkpoints and DNA repair.     

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.     

Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation

The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.     

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.