CDC5 Inhibits the Hyperphosphorylation of the Checkpoint Kinase Rad53, Leading to Checkpoint Adaptation

The Saccharomyces cerevisiae polo-like kinase Cdc5 promotes adaptation to the DNA damage checkpoint, in addition to its numerous roles in mitotic progression. The process of adaptation occurs when cells are presented with persistent or irreparable DNA damage and escape the cell-cycle arrest imposed by the DNA damage checkpoint. However, the precise mechanism of adaptation remains unknown. We report here that CDC5 is dose-dependent for adaptation and that its overexpression promotes faster adaptation, indicating that high levels of Cdc5 modulate the ability of the checkpoint to inhibit the downstream cell-cycle machinery. To pinpoint the step in the checkpoint pathway at which Cdc5 acts, we overexpressed CDC5 from the GAL1 promoter in damaged cells and examined key steps in checkpoint activation individually. Cdc5 overproduction appeared to have little effect on the early steps leading to Rad53 activation. The checkpoint sensors, Ddc1 (a member of the 9-1-1 complex) and Ddc2 (a member of the Ddc2/Mec1 complex), properly localized to damage sites. Mec1 appeared to be active, since the Rad9 adaptor retained its Mec1 phosphorylation. Moreover, the damage-induced interaction between phosphorylated Rad9 and Rad53 remained intact. In contrast, Rad53 hyperphosphorylation was significantly reduced, consistent with the observation that cell-cycle arrest is lost during adaptation. Thus, we conclude Cdc5 acts to attenuate the DNA damage checkpoint through loss of Rad53 hyperphosphorylation to allow cells to adapt to DNA damage. Polo-like kinase homologs have been shown to inhibit the ability of Claspin to facilitate the activation of downstream checkpoint kinases, suggesting that this function is conserved in vertebrates.     

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.     

Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

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Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein-protein interactions.     

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.     

Use of Mass Spectrometric Methods for Protein Identification in Receptor Research

Abstract

In recent years, mass spectrometry has become the method of choice for identifying small amounts of gel separated proteins. Using high mass accuracy peptide mass mapping followed if necessary by nanoelectrospray sequencing, most mammalian proteins can now be identified quickly and sensitively either in amino acid or in EST sequence databases. These methods are illustrated here using an ongoing project in the author's laboratory, a mass spectrometric screen for new mouse brain receptors and their interaction partners.     

Mass Spectrometric Analysis of the Antinociceptive Effect of Lidocaine

Relative measurements of the concentration of CO₂ released through the skin in rats in response to thermal stimulation were performed using a mass spectrometer with a membrane interface. It is demonstrated that the antinociceptive response to a pain stimulus during intraperitoneal propofol–lidocaine and propofol–ketamine anesthesia can be monitored using a mass spectrometer with a membrane interface. Lidocaine exerts direct action on the central nervous system and induces an antinociceptive effect in response to thermal stimulation.     

Reconstitution of Human Claspin-mediated Phosphorylation of Chk1 by the ATR (Ataxia Telangiectasia-mutated and Rad3-related) Checkpoint Kinase

ATR (ATM and Rad3-related) initiates a DNA damage signaling pathway in human cells upon DNA damage induced by UV and UV-mimetic agents and in response to inhibition of DNA replication. Genetic data with human cells and in vitro data with Xenopus egg extracts have led to the conclusion that the kinase activity of ATR toward the signal-transducing kinase Chk1 depends on the mediator protein Claspin. Here we have reconstituted a Claspin-mediated checkpoint system with purified human proteins. We find that the ATR-dependent phosphorylation of Chk1, but not p53, is strongly stimulated by Claspin. Similarly, DNA containing bulky base adducts stimulates ATR kinase activity, and Claspin acts synergistically with damaged DNA to increase phosphorylation of Chk1 by ATR. Mutations in putative phosphorylation sites in the Chk1-binding domain of Claspin abolish its ability to mediate ATR phosphorylation of Chk1. We also find that a fragment of Claspin containing the Chk1-binding domain together with a domain conserved in the yeast Mrc1 orthologs of Claspin is sufficient for its mediator activity. This in vitro system recapitulates essential components of the genetically defined ATR-signaling pathway.     

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.     

Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.     

快原子轰击质谱技术结合酶学方法鉴定黑曲霉1,6-缩水-β-D-吡喃葡萄糖激酶的诱导合成

1,6-缩水-β-D-吡喃葡萄糖是纤维素类物质热解的主要产物,黑曲霉突变株CBX209能较好地利用该糖作为唯一的碳源和能源生长并产生有用的代谢产物柠檬酸,其效率与利用葡萄糖大致相当。利用葡萄糖氧化酶和辣根过氧化物酶复合系统测定证明该菌株不存在1,6-缩水-β-D-吡喃葡萄糖水解酶。采用快原子轰击质谱技术结合6磷酸葡萄糖脱氢酶系统进行测定,结果表明经(NH₄)₂SO₄沉淀或阴离子交换层析处理后的无细胞提取液在加入ATP和Mg²⁺的条件下能直接催化1,6-缩水-β-D-吡喃葡萄糖合成6-磷酸葡萄糖,证明黑曲霉突变株中存在一个新酶,即1,6-缩水-β-D-吡喃葡萄糖激酶。该酶为诱导酶。     

快原子轰击质谱技术结合酶学方法鉴定黑曲霉1,6—缩水—β—D—吡喃葡萄糖激酶的诱导合成

1,6－缩水－β－D－吡喃葡萄糖是纤维素类物质热解的主要产物,黑曲霉突变株CBX－209能较好地利用该糖作为唯一的碳源和能源生长并产生有用的代谢产物柠檬酸,其效率与利用葡萄糖大致相当。利用葡萄糖氧化酶和竦根过氧化物酶复合系统测定证明该菌株不存在1,6－缩水－β－D－吡喃葡萄糖水解酶,采用快原子轰击质谱技术结合6－磷酸葡萄糖脱氢酶系统进行测定,结果表明经（NH4）2SO4沉淀或阴离子交换层析析处理后的无细胞提取液在加入ATP和Mg^2 条件下能直接催化1,6－缩水－β－D－吡喃葡萄糖合成6－磷酸葡萄糖,证明黑曲霉突变株中存在一个新酶,即1,6－缩水－β－D－吡喃葡萄糖激酶。该酶为诱导酶。     

Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome

Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use.     

Mass Spectrometric Identification of In Vivo Phosphorylation Sites of Differentially Expressed Proteins in Elongating Cotton Fiber Cells

Two-dimensional gel electrophoresis (2-DE)-based proteomics approach was applied to extensively explore the molecular basis of plant development and environmental adaptation. These proteomics analyses revealed thousands of differentially expressed proteins (DEPs) closely related to different biological processes. However, little attention has been paid to how peptide mass fingerprinting (PMF) data generated by the approach can be directly utilized for the determination of protein phosphorylation. Here, we used the software tool FindMod to predict the peptides that might carry the phosphorylation modification by examining their PMF data for mass differences between the empirical and theoretical peptides and then identified phosphorylation sites using MALDI TOF/TOF according to predicted peptide data from these DEP spots in the 2-D gels. As a result, a total of 48 phosphorylation sites of 40 DEPs were successfully identified among 235 known DEPs previously revealed in the 2-D gels of elongating cotton fiber cells. The 40 phosphorylated DEPs, including important enzymes such as enolase, transketolase and UDP-L-rhamnose synthase, are presumed to participate in the functional regulation of numerous metabolic pathways, suggesting the reverse phosphorylation of these proteins might play important roles in elongating cotton fibers. The results also indicated that some different isoforms of the identical DEP revealed in our 2-DE-based proteomics analysis could be annotated by phosphorylation events. Taken together, as the first report of large-scale identification of phosphorylation sites in elongating cotton fiber cells, our study provides not only an excellent example of directly identifying phosphorylation sites from known DEPs on 2-D gels but also provides a valuable resource for future functional studies of phosphorylated proteins in this field.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Autoinhibition and Autoactivation of the DNA Replication Checkpoint Kinase Cds1

Cds1 is the ortholog of Chk2 and the major effector of the DNA replication checkpoint in Schizosaccharomyces pombe. Previous studies have shown that Cds1 is activated by a two-stage mechanism. In the priming stage, the sensor kinase Rad3 and the mediator Mrc1 function to phosphorylate a threonine residue, Thr¹¹, in the SQ/TQ domain of Cds1. In the autoactivation stage, primed Cds1 molecules dimerize via intermolecular interactions between the phosphorylated Thr¹¹ in one Cds1 and the forkhead-associated domain of the other. Dimerization activates Cds1, probably by promoting autophosphorylation. To define the mechanisms for the autoactivation of primed Cds1 and the regulation of this process, we carried out genetic and biochemical studies to identify phosphorylatable residues required for checkpoint activation. Our data indicate that dimerization of Cds1 promotes trans-autophosphorylation of a number of residues in the catalytic domain, but phosphorylation of a highly conserved threonine residue (Thr³²⁸) in the activation loop is the only covalent modification required for kinase activation in vitro and in vivo. Autophosphorylation of Thr³²⁸ and kinase activation in unprimed, monomeric Cds1 are strongly inhibited by the C-terminal 27-amino acid tail of the enzyme. This autoinhibitory effect may play an important role in preventing spontaneous activation of the replication checkpoint during normal cell cycles. The two-stage activation pathway and the autoinhibition mechanism, which are probably shared by other members of the Chk2 family, provide sensitivity, specificity, and noise immunity, properties required for the replication checkpoint.DNA replication forks can be arrested or stalled by damage to DNA templates, depletion of deoxyribonucleotides, or inhibition of replisome enzymes (1). If undetected, arrested or stalled replication forks may undergo collapse, resulting in loss of genetic information, mutagenesis, or even cell death. To maintain the integrity of the genome, eukaryotes have evolved a surveillance mechanism called the “replication checkpoint” that can detect perturbations of DNA replication and elicit a number of cellular responses that serve to mitigate the effects of such perturbations. These cellular responses may include stabilization of replication forks, suppression of initiation of DNA replication, increased DNA repair activity, augmented production of deoxyribonucleotide precursors, and delay of mitosis. The replication checkpoint pathway is essential for cell survival under a variety of stressful conditions and has been conserved from yeast to humans (for reviews, see Refs. 1–3). Mutations in the pathway are also linked to cancer (4–6).The replication checkpoint is a complex signal transduction pathway that can be separated conceptually into three functional components. Sensors detect the perturbed DNA replication forks; mediators transduce the checkpoint signal, whereas effectors regulate the cell cycle and promote cell survival. Genetic studies, especially those in the yeasts, have identified most, if not all, of the essential factors of the pathway. In the fission yeast Schizosaccharomyces pombe, six Rad proteins mediate the sensor function (for reviews, see Refs. 7 and 8). The protein kinase Rad3 (ATR in human cells) binds an essential co-factor Rad26 (ATRIP in human cells), and the complex associates with stalled replication forks. Rad9, Hus1, and Rad1 form the “9-1-1” ring structure similar to that of the replication processivity factor proliferating cell nuclear antigen. Rad17, in association with Rfc2-5, loads the 9-1-1 complex onto DNA at stalled forks. After detection of stalled forks by the sensor complexes, the mediator protein Mrc1 protein (Claspin in human cells) functions to facilitate the Rad3-dependent phosphorylation and activation of the effector protein kinase Cds1 (Chk2 in human cells) (9–11). Studies in Saccharomyces cerevisiae suggest that Mrc1 may be a component of the replisome (12, 13). A second mediator, Crb2 (BRCA1 in human cells) (14, 15), functions in response to DNA damage either within or outside of S phase. Crb2 facilitates the activation of a second effector kinase, Chk1.We have previously reported that in S. pombe, the effector kinase of the replication checkpoint pathway, Cds1, is activated by a two-stage mechanism (11). In the first or priming stage, the sensor kinase Rad3 phosphorylates two functionally redundant Cds1-docking repeats in the middle of the mediator Mrc1. The phosphorylated docking repeats on Mrc1 recruit Cds1 to the stalled replication fork by a phospho-dependent interaction with the forkhead-associated (FHA)3 domain of Cds1. Once recruited to the proximity of the assembled sensor complex, Cds1 is phosphorylated by Rad3 at Thr¹¹. In the second or autoactivation stage, primed Cds1 molecules dimerize by two identical intermolecular interactions between phosphorylated Thr¹¹ and the FHA domain. Dimerization promotes autophosphorylation and activation of Cds1. This two-stage activation mechanism is supported by genetic studies (9, 16–18) and is probably similar to the activation pathway for mammalian Chk2 (19–23). Although many steps in the pathway are now understood, the precise biochemical mechanism of autoactivation of primed Cds1 has not been well defined.Protein kinases can be activated by a variety of mechanisms. Although phosphorylation of the activation loop, usually by an upstream kinase of a signal transduction pathway, is the most common mechanism for kinase activation, some protein kinases can be activated by phosphorylation of residues outside the activation loop (for reviews, see Refs. 24 and 25). Other protein kinases can be activated without phosphorylation (e.g. by intermolecular interactions following dimerization) (26), by removal of an inhibitory element (27), or by binding to an activator (27, 28). Since the autoactivation of primed Cds1 requires dimerization, three possible activation mechanisms can be proposed. First, like many other protein kinases, Cds1 may be activated by phosphorylation of the activation loop (24). There are several known examples of kinase activation via trans-autophosphorylation of the activation loop. In these cases, the activation loop usually contains a consensus phosphorylation site of the kinase itself. This is not the case for Cds1 family kinases. A second possibility is that dimerization of Cds1 may allow intermolecular interactions that promote activation, as has been suggested for the epidermal growth factor receptor (26). Finally, activation of Cds1 may be a consequence of phosphorylation of residue(s) outside the activation loop. In the second and the third models, phosphorylation of the two essential threonine residues in the activation segment observed previously in mammalian Chk2 (22) and in the S. cerevisiae homologue Rad53 (29) would be a by-product, not a cause, of kinase activation.Several previous observations have provided evidence in support of the possibility that activation of Cds1 requires autophosphorylation. First, Cds1 is a phosphoprotein, and hydroxyurea (HU) treatment of cells induces further phosphorylation that is partially dependent on the kinase activity of Cds1 itself.⁴ In the case of mammalian Chk2, the ortholog of Cds1, sites of phosphorylation have been mapped to the activation segment residues, Thr³⁸³ and Thr³⁸⁷ (22, 30), as well as to residues Ser³⁷⁹ (31), Ser⁵¹⁶ (30, 32), and Ser⁴⁵⁶ (33), which lie outside of the activation segment. Phosphorylation has also been mapped by mass spectrometry to sites within and outside of the activation segment of Rad53 (29), the S. cerevisiae homologue of Cds1. Second, genetic studies have shown that residues Thr³²⁸ and Thr³³² in the activation segment of Cds1 (corresponding to Thr³⁸³ and Thr³⁸⁷ of Chk2 and Thr³⁵⁴ and Thr³⁵⁸ of Rad53) are essential for kinase activity (11, 34). Third, phosphatase treatment of “activated Cds1” purified from HU-treated cells abolishes kinase activity (11). Finally, activation of induced Cds1 dimers in vitro is dependent upon ATP (11).In this report, we describe experiments aimed at distinguishing among the three potential mechanisms for Cds1 activation. We show that there are only three phosphorylatable residues in the Cds1 kinase domain (Thr³²⁸, Thr³³², and Tyr³⁵²) that are essential for activation of the replication checkpoint in vivo and for enzyme activity in vitro. Of these three residues, Thr³²⁸ in the activation loop is a target of autophosphorylation, and its phosphorylation is the only covalent modification required for Cds1 activation. Autophosphorylation of Thr³²⁸ occurs in trans and only proceeds at an appreciable rate when the enzyme is at high local concentration. Presumably, one molecule in a Cds1 dimer transiently assumes an active conformation and phosphorylates the Thr³²⁸ in the activation loop of the other molecule. The activated molecule can then rapidly phosphorylate its dimeric partner. The second essential residue, Thr³³², which is also in the activation loop, is not phosphorylated and is likely required, directly or indirectly, for catalysis. The third essential residue Tyr³⁵² can be autophosphorylated in vitro with the Cds1 purified from S. pombe, and its phosphorylation is strongly stimulated by dimerization. However, Tyr³⁵² phosphorylation is not readily observed in vivo and is not required for Cds1 activation. Our data rule out the other two possible mechanisms for Cds1 activation: phosphorylation of sites outside of the activation segment and phosphorylation-independent conformational changes induced by dimerization. We also report that the C terminus of Cds1 is a cis-regulatory element that can dramatically suppress Cds1 autoactivation in vitro and in vivo. Taken together, our data explain how the replication checkpoint can be sensitive and specific and also possess a high threshold for spontaneous activation.     

检控蛋白Rad17在小鼠睾丸精子发生过程中的表达及其磷酸化变化

Rad17是细胞周期检控点信号转导过程中的一个关键检控蛋白,在DNA损伤检控和DNA复制检控中具有重要功能。但Rad17在细胞减数分裂中的检控作用还不是很清楚。因细胞减数分裂在睾丸组织中非常活跃,应用Western印迹检测Rad17在不同发育时期的小鼠睾丸组织中的表达及其磷酸化水平,并应用免疫组化的方法检测小鼠睾丸组织不同时期生殖细胞内Rad17的表达变化。结果显示Rad17在小鼠睾丸组织内高表达,而在肝、肾等组织中表达水平较低;Rad17在不同周龄的小鼠睾丸组织中均高水平表达,但在4周龄以后的小鼠睾丸组织中其磷酸化水平明显升高;免疫组化结果显示Rad17在精原细胞、精母细胞的细胞核中高表达,但在成熟精子细胞中消失。这些结果提示Rad17在小鼠睾丸生殖细胞减数分裂过程中也起重要检控作用。