C. elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein.

BACKGROUND: In response to genotoxic stress, cells activate checkpoint pathways that lead to a transient cell cycle arrest that allows for DNA repair or to apoptosis, which triggers the demise of genetically damaged cells. RESULTS: During positional cloning of the C. elegans rad-5 DNA damage checkpoint gene, we found, surprisingly, that rad-5(mn159) is allelic with clk-2(qm37), a mutant previously implicated in regulation of biological rhythms and life span. However, clk-2(qm37) is the only C. elegans clock mutant that is defective for the DNA damage checkpoint. We show that rad-5/clk-2 acts in a pathway that partially overlaps with the conserved C. elegans mrt-2/S. cerevisiae RAD17/S. pombe rad1(+) checkpoint pathway. In addition, rad-5/clk-2 also regulates the S phase replication checkpoint in C. elegans. Positional cloning reveals that the RAD-5/CLK-2 DNA damage checkpoint protein is homologous to S. cerevisiae Tel2p, an essential DNA binding protein that regulates telomere length in yeast. However, the partial loss-of-function C. elegans rad-5(mn159) and clk-2(qm37) checkpoint mutations have little effect on telomere length, and analysis of the partial loss-of-function of S. cerevisiae tel2-1 mutant failed to reveal typical DNA damage checkpoint defects. CONCLUSIONS: Using C. elegans genetics we define the novel DNA damage checkpoint protein RAD-5/CLK-2, which may play a role in oncogenesis. Given that Tel2p has been shown to bind to a variety of nucleic acid structures in vitro, we speculate that the RAD-5/CLK-2 checkpoint protein may act at sites of DNA damage, either as a sensor of DNA damage or to aid in the repair of damaged DNA.     

C. elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein

Background: In response to genotoxic stress, cells activate checkpoint pathways that lead to a transient cell cycle arrest that allows for DNA repair or to apoptosis, which triggers the demise of genetically damaged cells.Results: During positional cloning of the C. elegans rad-5 DNA damage checkpoint gene, we found, surprisingly, that rad-5(mn159) is allelic with clk-2(qm37), a mutant previously implicated in regulation of biological rhythms and life span. However, clk-2(qm37) is the only C. elegans clock mutant that is defective for the DNA damage checkpoint. We show that rad-5/clk-2 acts in a pathway that partially overlaps with the conserved C. elegans mrt-2/S. cerevisiae RAD17/S. pombe rad1(+) checkpoint pathway. In addition, rad-5/clk-2 also regulates the S phase replication checkpoint in C. elegans. Positional cloning reveals that the RAD-5/CLK-2 DNA damage checkpoint protein is homologous to S. cerevisiae Tel2p, an essential DNA binding protein that regulates telomere length in yeast. However, the partial loss-of-function C. elegans rad-5(mn159) and clk-2(qm37) checkpoint mutations have little effect on telomere length, and analysis of the partial loss-of-function of S. cerevisiae tel2-1 mutant failed to reveal typical DNA damage checkpoint defects.Conclusions: Using C. elegans genetics we define the novel DNA damage checkpoint protein RAD-5/CLK-2, which may play a role in oncogenesis. Given that Tel2p has been shown to bind to a variety of nucleic acid structures in vitro, we speculate that the RAD-5/CLK-2 checkpoint protein may act at sites of DNA damage, either as a sensor of DNA damage or to aid in the repair of damaged DNA.     

C. elegans clk-2, a gene that limits life span, encodes a telomere length regulator similar to yeast telomere binding protein Tel2p.

An important quest in modern biology is to identify genes involved in aging. Model organisms such as the nematode Caenorhabditis elegans are particularly useful in this regard. The C. elegans genome has been sequenced [1], and single gene mutations that extend adult life span have been identified [2]. Among these longevity-controlling loci are four apparently unrelated genes that belong to the clk family. In mammals, telomere length and structure can influence cellular, and possibly organismal, aging. Here, we show that clk-2 encodes a regulator of telomere length in C. elegans.     

Sensitivity of Caenorhabditis elegans clk-1 mutants to ubiquinone side-chain length reveals multiple ubiquinone-dependent processes

Ubiquinone (coenzyme Q, or Q) is a membrane constituent, whose head group is capable of accepting and donating electrons and whose lipidic side chain is composed of a variable number of isoprene subunits. A possible role for Q as a dietary antioxidant for treating conditions that involve altered cellular redox states is being intensely studied. Mutations in the clk-1 gene of the nematode Caenorhabditis elegans affect numerous physiological rates including behavioral rates, developmental rates, reproduction, and life span. clk-1 encodes a protein associated with the inner mitochondrial membrane that is necessary for Q biosynthesis in C. elegans. clk-1 mutants do not synthesize Q but accumulate demethoxyubiquinone, a Q synthesis intermediate that is able to partially sustain mitochondrial respiration in worms as well as in mammals. Recently, we and others have found that exogenous Q is necessary for the fertility and development of clk-1 mutants. Here, we take advantage of the clk-1 genetic model to identify structural features of Q that are functionally important in vivo. We show that clk-1 mutants are exquisitely sensitive to the length of the side chain of the Q they consume. We also identified differential sensitivity to Q side-chain length between null alleles of clk-1 (qm30 and qm51) and the weaker allele e2519. This allows us to propose a model where we distinguish several types of Q-dependent processes in vivo: processes that are very sensitive to Q side-chain length and processes that are permissive to Q with shorter chains.     

Human CLK2 links cell cycle progression,apoptosis, and telomere length regulation

Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form.     

Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.     

Phenotypic and suppressor analysis of defecation in clk-1 mutants reveals that reaction to changes in temperature is an active process in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans maternal-effect gene clk-1 affect cellular, developmental, and behavioral timing. They result in a slowing of the cell cycle, embryonic and postembryonic development, reproduction, and aging, as well as of the defecation, swimming, and pharyngeal pumping cycles. Here, we analyze the defecation behavior in clk-1 mutants, phenotypically and genetically. When wild-type worms are grown at 20 degrees and shifted to a new temperature, the defecation cycle length is significantly affected by that new temperature. In contrast, we find that when clk-1 mutants are shifted, the defecation cycle length is unaffected by that new temperature. We carried out a screen for mutations that suppress the slow defecation phenotype at 20 degrees and identified two distinct classes of genes, which we call dsc for defecation suppressor of clk-1. Mutations in one class also restore the ability to react normally to changes in temperature, while mutations in the other class do not. Together, these results suggest that clk-1 is necessary for readjusting the defecation cycle length in response to changes in temperature. On the other hand, in the absence of clk-1 activity, we observe temperature compensation, a mechanism that maintains a constant defecation period in the face of changes in temperature.     

clk-1, mitochondria, and physiological rates

Mutations in the C. elegans maternal-effect gene clk-1 are highly pleiotropic, affecting the duration of diverse developmental and behavioral processes. They result in an average slowing of embryonic and post-embryonic development, adult rhythmic behaviors, reproduction, and aging.(1) CLK-1 is a highly conserved mitochondrial protein,(2,3) but even severe clk-1 mutations affect mitochondrial respiration only slightly.(3) Here, we review the evidence supporting the regulatory role of clk-1 in physiological timing. We also discuss possible models for the action of CLK-1, in particular, one proposing that CLK-1 is involved in the coordination of mitochondrial and nuclear function. BioEssays 22:48-56, 2000.     

Molecular mechanism of maternal rescue in the clk-1 mutants of Caenorhabditis elegans

The clk-1 mutants of Caenorhabditis elegans display an average slowing down of physiological rates, including those of development, various behaviors, and aging. clk-1 encodes a hydroxylase involved in the biosynthesis of the redox-active lipid ubiquinone (co-enzyme Q), and in clk-1 mutants, ubiquinone is replaced by its biosynthetic precursor demethoxyubiquinone. Surprisingly, homozygous clk-1 mutants display a wild-type phenotype when issued from a heterozygous mother. Here, we show that this maternal effect is the result of the persistence of small amounts of maternally derived CLK-1 protein and that maternal CLK-1 is sufficient for the synthesis of considerable amounts of ubiquinone during development. However, gradual depletion of CLK-1 and ubiquinone, and expression of the mutant phenotype, can be produced experimentally by developmental arrest. We also show that the very long lifespan observed in daf-2 clk-1 double mutants is not abolished by the maternal effect. This suggests that, like developmental arrest, the increased lifespan conferred by daf-2 allows for depletion of maternal CLK-1, resulting in the expression of the synergism between clk-1 and daf-2. Thus, increased adult longevity can be uncoupled from the early mutant phenotypes, indicating that it is possible to obtain an increased adult lifespan from the late inactivation of processes required for normal development and reproduction.     

Mutations in the Clk-1 Gene of Caenorhabditis Elegans Affect Developmental and Behavioral Timing

We have identified three allelic, maternal-effect mutations that affect developmental and behavioral timing in Caenorhabditis elegans. They result in a mean lengthening of embryonic and postembryonic development, the cell cycle period and life span, as well as the periods of the defecation, swimming and pumping cycles. These mutants also display a number of additional phenotypes related to timing. For example, the variability in the length of embryonic development is several times larger in the mutants than in the wild type, resulting in the occasional production of mutant embryos developing more rapidly than the most rapidly developing wild-type embryos. In addition, the duration of embryonic development of the mutants, but not of the wild type, depends on the temperature at which their parents were raised. Finally, individual variations in the severity of distinct mutant phenotypes are correlated in a counterintuitive way. For example, the animals with the shortest embryonic development have the longest defecation cycle and those with the longest embryonic development have the shortest defecation cycle. Most of the features affected by these mutations are believed to be controlled by biological clocks, and we therefore call the gene defined by these mutations clk-1, for ``abnormal function of biological clocks.'     

The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA.

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.     

Mitochondrial oxidative stress alters a pathway in Caenorhabditis elegans strongly resembling that of bile acid biosynthesis and secretion in vertebrates

Mammalian bile acids (BAs) are oxidized metabolites of cholesterol whose amphiphilic properties serve in lipid and cholesterol uptake. BAs also act as hormone-like substances that regulate metabolism. The Caenorhabditis elegans clk-1 mutants sustain elevated mitochondrial oxidative stress and display a slow defecation phenotype that is sensitive to the level of dietary cholesterol. We found that: 1) The defecation phenotype of clk-1 mutants is suppressed by mutations in tat-2 identified in a previous unbiased screen for suppressors of clk-1. TAT-2 is homologous to ATP8B1, a flippase required for normal BA secretion in mammals. 2) The phenotype is suppressed by cholestyramine, a resin that binds BAs. 3) The phenotype is suppressed by the knock-down of C. elegans homologues of BA-biosynthetic enzymes. 4) The phenotype is enhanced by treatment with BAs. 5) Lipid extracts from C. elegans contain an activity that mimics the effect of BAs on clk-1, and the activity is more abundant in clk-1 extracts. 6) clk-1 and clk-1;tat-2 double mutants show altered cholesterol content. 7) The clk-1 phenotype is enhanced by high dietary cholesterol and this requires TAT-2. 8) Suppression of clk-1 by tat-2 is rescued by BAs, and this requires dietary cholesterol. 9) The clk-1 phenotype, including the level of activity in lipid extracts, is suppressed by antioxidants and enhanced by depletion of mitochondrial superoxide dismutases. These observations suggest that C. elegans synthesizes and secretes molecules with properties and functions resembling those of BAs. These molecules act in cholesterol uptake, and their level of synthesis is up-regulated by mitochondrial oxidative stress. Future investigations should reveal whether these molecules are in fact BAs, which would suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome might participate in disease processes by affecting the regulation of metabolism by BAs.     

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.     

Amino acid changes in Xrs2p,Dun1p,and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.     

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.     

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cerevisiae and lacks antioxidant activity

Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q(9) and instead accumulate demethoxy-Q(9) (DMQ(9)). DMQ(9) has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ(6) in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q(6) and producing solely DMQ were respiratory deficient and unable to support (6)either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ(6) failed to protect cells against oxidative stress generated by H(2)O(2) or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.     

C. elegans knockouts in ubiquinone biosynthesis genes result in different phenotypes during larval development

Ubiquinone is an essential molecule in aerobic organisms to achieve both, ATP synthesis and antioxidant defence. Mutants in genes responsible of ubiquinone biosynthesis lead to non-respiring petite yeast. In C. elegans, coq-7/clk-1 but not coq-3 mutants live longer than wild type showing a 'slowed' phenotype. In this paper we demonstrate that absence in ubiquinone in coq-1, coq-2 or coq-8 mutants lead to larval development arrest, slowed pharyngeal pumping, eventual paralysis and cell death. All these features emerge during larval development, whereas embryo development appeared similar to that of wild type individuals. Dietary coenzyme Q did not restore any of the alterations found in these coq mutants. These phenomena suggest that coenzyme Q mutants unable to synthesize this molecule develop a deleterious phenotype leading to lethality. On the contrary, phenotype of C. elegans coq-7/clk-1 mutants may be a unique phenotype than can not generalize to mutants in ubiquinone biosynthesis. This particular phenotype may not be based on the absence of endogenous coenzyme Q, but to the simultaneous presence of dietary coenzyme Q and the its biosynthesis intermediate demethoxy-coenzyme Q.     

S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association

In diverse organisms, the Mre11 complex and phosphoinositide 3-kinase-related kinases (PIKKs), such as Tel1p and Mec1p from S. cerevisiae, are key mediators of DNA repair and DNA damage checkpoints that also function at telomeres. Here, we use chromatin immunoprecipitation (ChIP) to determine if Mre11p, Tel1p, or Mec1p affects telomere maintenance by promoting recruitment of telomerase subunits to S. cerevisiae telomeres. We find that recruitment of Est2p, the catalytic subunit of telomerase, and Est1p, a telomerase accessory protein, was severely reduced in mre11Delta and tel1Delta cells. In contrast, the levels of Est2p and Est1p binding in late S/G2 phase, the period in the cell cycle when yeast telomerase lengthens telomeres, were indistinguishable in wild-type (WT) and mec1Delta cells. These data argue that Mre11p and Tel1p affect telomere length by promoting telomerase recruitment to telomeres, whereas Mec1p has only a minor role in telomerase recruitment in a TEL1 cell.