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• W2059182662 abstract "The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks. The current model is that after DNA damage or replication blocks, ATRMec1 phosphorylates Chk1 on the non-catalytic C-terminal domain. However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus in vivo remains largely unknown. In this study we used an in vivo assay to examine the role of the C terminus of Chk1 in the response to DNA damage and replication blocks. The conserved ATRMec1 phosphorylation sites were essential for the checkpoint response to DNA damage and replication blocks in vivo; that is, that mutation of the sites caused lethality when DNA replication was stalled by hydroxyurea. Despite this, loss of the ATRMec1 phosphorylation sites did not change the kinase activity of Chk1 in vitro. Furthermore, a single amino acid substitution at an invariant leucine in a conserved domain of the non-catalytic C terminus restored viability to cells expressing the ATRMec1 phosphorylation site-mutated protein and relieved the requirement of an upstream mediator for Chk1 activation. Our findings show that a single amino acid substitution in the C terminus, which could lead to an allosteric change in Chk1, allows it to bypass the requirement of the conserved ATRMec1 phosphorylation sites for checkpoint function. The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks. The current model is that after DNA damage or replication blocks, ATRMec1 phosphorylates Chk1 on the non-catalytic C-terminal domain. However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus in vivo remains largely unknown. In this study we used an in vivo assay to examine the role of the C terminus of Chk1 in the response to DNA damage and replication blocks. The conserved ATRMec1 phosphorylation sites were essential for the checkpoint response to DNA damage and replication blocks in vivo; that is, that mutation of the sites caused lethality when DNA replication was stalled by hydroxyurea. Despite this, loss of the ATRMec1 phosphorylation sites did not change the kinase activity of Chk1 in vitro. Furthermore, a single amino acid substitution at an invariant leucine in a conserved domain of the non-catalytic C terminus restored viability to cells expressing the ATRMec1 phosphorylation site-mutated protein and relieved the requirement of an upstream mediator for Chk1 activation. Our findings show that a single amino acid substitution in the C terminus, which could lead to an allosteric change in Chk1, allows it to bypass the requirement of the conserved ATRMec1 phosphorylation sites for checkpoint function. Cell cycle checkpoints coordinate the maintenance of genomic integrity with cell division. The checkpoints that monitor replication fork integrity and DNA damage lesions sensed outside of DNA replication use similar mechanisms, and sometimes some of the same proteins, to delay cell cycle transitions, and in addition play an important role in the stability of replication forks and in DNA repair (1Nyberg K.A. Michelson R.J. Putnam C.W. Weinert T.A. Annu. Rev. Genet. 2002; 36: 617-656Crossref PubMed Scopus (647) Google Scholar, 2Branzei D. Foiani M. Curr. Opin. Cell Biol. 2005; 17: 568-575Crossref PubMed Scopus (195) Google Scholar).The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks in metazoans. Chk1 is activated in budding yeast when damage is sensed outside the context of a replication fork. In this case the checkpoint proteins in addition to the kinase Chk1 and the mediator/signal amplifier protein Rad9 inhibit spindle elongation and chromosome segregation in response to DNA damage sensed in late S or G2. One effector of Chk1 in this response is the securin Pds1, which is stabilized by phosphorylation in response to DNA damage (3Gardner R. Putnam C.W. Weinert T. EMBO J. 1999; 18: 3173-3185Crossref PubMed Scopus (144) Google Scholar, 4Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (667) Google Scholar, 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar, 6Searle J.S. Schollaert K.L. Wilkins B.J. Sanchez Y. Nat. Cell Biol. 2004; 6: 138-145Crossref PubMed Scopus (77) Google Scholar). Although the Chk1/Rad9 DNA damage checkpoint arm is not required during replication blocks in the budding yeast, we have shown that Chk1 is activated when replication is slowed down by the ribonucleotide reductase inhibitor hydroxyurea (HU). 3The abbreviations used are: HU, hydroxyurea; HA, hemagglutinin; BD, binding domain; AD, activation domain; SpChk1, S. pombe Chk1; MMS, methyl methanesulfonate; WT, wild type; 3AQ, A333Q, A356Q, and A382Q.3The abbreviations used are: HU, hydroxyurea; HA, hemagglutinin; BD, binding domain; AD, activation domain; SpChk1, S. pombe Chk1; MMS, methyl methanesulfonate; WT, wild type; 3AQ, A333Q, A356Q, and A382Q. We have also shown that cells lacking the checkpoint kinase Dun1 are dependent on the Chk1/Rad9 arm for survival of replication blocks elicited by HU (7Schollaert K. Poisson J.M. Searle J.S. Scwanekamp J.A. Tomlinson C.R. Sanchez Y. Mol. Biol. Cell. 2004; 15: 4051-4063Crossref PubMed Google Scholar, 8Caldwell J.M. Chen Y. Schollaert K. Theis J.F. Babcock G. Newlon C.S. Sanchez Y. J. Cell Biol. 2008; 180: 1073-1086Crossref PubMed Scopus (7) Google Scholar). Studies addressing the in vivo roles of mammalian Chk1 have been difficult due to the fact that Chk1 is essential for early development of mouse embryos (9Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (192) Google Scholar). Therefore, yeast cells lacking Dun1 provide a valuable model to explore the role of Chk1 in vivo in the recovery from replication blocks and the response to DNA damage generated outside of S phase.The Chk1 proteins consist of two primary domains, the well conserved N-terminal kinase domain and the less conserved non-catalytic C-terminal domain (10Palermo C. Hope J.C. Freyer G.A. Rao H. Walworth N.C. PLoS ONE. 2008; 3: e1427Crossref PubMed Scopus (14) Google Scholar, 11Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1117) Google Scholar). The C-terminal domains of the Chk1 orthologues are phosphorylated after DNA damage. The C-terminal domain of vertebrate Chk1 has also been suggested to play an inhibitory role in the kinase activity of the protein (12Katsuragi Y. Sagata N. Mol. Biol. Cell. 2004; 15: 1680-1689Crossref PubMed Scopus (61) Google Scholar). Kinases are regulated by allosteric changes caused by post-translational modifications, by protein interactions (mitogen-activated protein kinases and protein kinase A (PKA)), by modulating access to substrates via subcellular localization (IκB kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)), and by binding of inhibitors (cyclin-dependent kinases, PKA) (13Cheetham G.M. Curr. Opin. Struct. Biol. 2004; 14: 700-705Crossref PubMed Scopus (51) Google Scholar, 14Pellicena P. Kuriyan J. Curr. Opin. Struct. Biol. 2006; 16: 702-709Crossref PubMed Scopus (86) Google Scholar, 15Das R. Esposito V. Abu-Abed M. Anand G.S. Taylor S.S. Melacini G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 93-98Crossref PubMed Scopus (108) Google Scholar). However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus remains largely unknown.We used our in vivo assay in the genetically tractable model system (Saccharomyces cerevisiae) and cells lacking Dun1 to examine the role of Chk1 in the response to DNA damage and replication blocks. Using this assay we explored the role of phosphorylation and conserved domains of Chk1 in its regulation. We found that the conserved ATRMec1 phosphorylation sites (equivalent to Ser-317 and Ser-345 in human Chk1) of Chk1 are required for both the response to DNA damage and replication blocks. We also identified residues on the same face of a predicted α-helix in the GD domain of the C terminus of Chk1 that when changed result in a kinase that is constitutively modified in the absence of a checkpoint signal. In particular, a single amino acid substitution at a conserved leucine in the GD domain resulted in a constitutively active kinase that did not require the conserved ATR phosphorylation sites of Chk1 for checkpoint activation and function.EXPERIMENTAL PROCEDURESChk1 Mutant Alleles—All mutant chk1 alleles were constructed by sequential PCR. The template for both reactions was a plasmid containing the wild-type CHK1. First, a PCR was carried out using a primer containing the desired mutation and a primer upstream of the BamHI in CHK1. This PCR product was then used as a “mega primer” along with a primer from the polylinker for the second PCR. The product of the second PCR was digested with BamHI and SacI and used to replace the corresponding fragment in pML107.1 (5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar), which carries an HA-tagged wild-type CHK1. All mutations were confirmed by sequencing.Yeast Strains—The yeast strains used in this study are listed in Table 1. Strains were generated using standard genetic techniques. Integration of mutant chk1 alleles in cdc13-1 or dun1Δ cells was accomplished by cloning the C-terminal PshAI-SacI fragment of the chk1 alleles into integrating vector pRS406 (16Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) digested with SmaI-SacI. The resulting partial chk1 constructs were linearized by MfeI and then transformed into a cdc13-1 or dun1Δ strain. Plasmid integration resulted in a genetic exchange at the CHK1 locus consisting of a full-length mutant chk1 allele and a 3′-truncated (nonfunctional) copy of the endogenous wild-type allele. The expected genome changes were confirmed by diagnostic PCR followed by restriction enzyme digestion or sequencing.TABLE 1Yeast strains used in this studyStrainsGenotypeSourceY300MATa ade2-1 trp1-1 ura3-1 leu2-3,112 his 3-11,15 can1-100Ref. 26Allen J.B. Zhou Z. Siede W. Friedberg E.C. Elledge S.J. Genes Dev. 1994; 8: 2401-2415Crossref PubMed Scopus (343) Google ScholarY801As Y300 chk1Δ::HIS3Ref. 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google ScholarYJP8As Y300 chk1Δ::HIS3 dun1Δ::HIS3Ref. 7Schollaert K. Poisson J.M. Searle J.S. Scwanekamp J.A. Tomlinson C.R. Sanchez Y. Mol. Biol. Cell. 2004; 15: 4051-4063Crossref PubMed Google ScholarY578As Y300 dun1Δ::HIS3Ref. 27Desany B.A. Alcasabas A.A. Bachant J.B. Elledge S.J. Genes Dev. 1998; 12: 2956-2970Crossref PubMed Scopus (382) Google ScholarYHC300As Y578 chk1Δ::URA3This studyYHC302rAs Y578 chk1-ΔGD::URA3This studyYHC305As Y578 chk1-G503D::URA3This studyYHC306As Y578 chk1-L506R::URA3This studyYHC308As Y578 chk1-ΔTKF::URA3This studyYHC312As Y578 chk1-F419A::URA3This studyYHC332As Y578 chk1-3AQ::URA3This studyYHC334As Y578 chk1-L506R, 3AQ::URA3This studyYHC335As Y578 chk1-T421A::URA3This studyYHC336As Y578 chk1-L506R, T421A::URA3This studyYJP321As Y578 rad9Δ::HIS3Ref. 7Schollaert K. Poisson J.M. Searle J.S. Scwanekamp J.A. Tomlinson C.R. Sanchez Y. Mol. Biol. Cell. 2004; 15: 4051-4063Crossref PubMed Google ScholarYJP1129As Y578 chk1-L506R::URA3 rad9Δ::HIS3This studyYJP1130As Y578 chk1-L506R, 3AQ::URA3 rad9Δ::HIS3This studyY438As Y300 rad9Δ::HIS3Ref. 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google ScholarYJP219As Y300 chk1Δ::URA3 rad9Δ::HIS3This studyY581As Y300 mec1Δ::HIS3 trp1-1::GAP-RNR1-TRP1Ref. 27Desany B.A. Alcasabas A.A. Bachant J.B. Elledge S.J. Genes Dev. 1998; 12: 2956-2970Crossref PubMed Scopus (382) Google ScholarY816As Y300 cdc13-1Ref. 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google ScholarY818As Y300 cdc13-1 chk1Δ::HIS3Ref. 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google ScholarY811As Y300 cdc13-1 chk1Δ::HIS3 PDS1-3XHA::URA3Ref. 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google ScholarYHC200As Y816 chk1Δ::URA3This studyYHC202As Y816 chk1-ΔGD::URA3This studyYHC205As Y816 chk1-G503D::URA3This studyYHC206As Y816 chk1-L506R::URA3This studyYHC208As Y816 chk1-ΔTKF::URA3This studyYHC212As Y816 chk1-F419A::URA3This studyYHC232As Y816 chk1-3AQ::URA3This studyYHC234As Y816 chk1-L506R, 3AQ::URA3This studyYJP1124As Y816 rad9Δ::HIS3This studyYJP1159As Y816 chk1-L506R::URA3 rad9Δ::HIS3This studyYJP1160As Y816 chk1-L506R, 3AQ::URA3 rad9Δ::HIS3This studyPJ69-4AMATa trpl-901 leu2-3,112 ura3-52 his3-200 ga14Δ ga18Δ LYSZ::GALl-HIS3 GAL2-ADE2 metZ::GAL7-lacZRef. 17James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar Open table in a new tab Growth Conditions—Yeast cells were grown on yeast extract/peptone/dextrose-rich medium or synthetic complete medium as indicated. To examine sensitivity to HU on plates, serial dilutions of cells were spotted on to yeast extract/peptone/dextrose solid medium containing HU ranging in concentration from 0 to 80 mm, incubated at 30 °C for 3 days, and then photographed. The micro-colony assays were conducted by growing cells in yeast extract/peptone/dextrose at 22 °C overnight and then diluted and plated on prewarmed plates and incubated at 30 °C. After 12–14 h, cells were examined for formation of microcolonies, and the number of cells in each of 50–100 microcolonies was counted for each strain. For Western analysis of the Chk1 shift caused by DNA damage, cells were grown in synthetic complete–Leu medium at 30 °C to log phase, and MMS was added to a final concentration of 0.1%. Cells were then incubated at 30 °C for 2 h before harvest.Western Analysis—Protein extracts from cells expressing HA-CHK1 and 3XHA-PDS1 were prepared as previously described (25Foiani M. Marini F. Gamba D. Lucchini G. Plevani P. Mol. Cell. Biol. 1994; 14: 923-933Crossref PubMed Google Scholar), separated on 10% acrylamide, 0.066% bisacrylamide gels, and transferred to nitrocellulose membranes. HA-Chk1 and HA-Pds1 were detected by Western analysis using anti-HA antibody (16B12, Covance, Madison, WI).Two-hybrid Assay—The two-hybrid vectors and host strain of James et al. (17James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) were used. The CHK1 coding region and its mutant forms were inserted into pGBD-C1 to produce Chk1-Gal4 DNA binding domain (BD) fusion proteins, whereas the RAD9 coding region was inserted into pGAD-C1 to yield a Rad9-Gal4 activation domain (AD) fusion protein. Chk1-BD and Rad9-AD plasmids were co-transformed into strain PJ69-4A, which carries a GAL2-ADE2 reporter gene. Transformants were picked and suspended in media with a starting cell concentration of 2 × 107/ml. A series of 10-fold dilutions were made, and five μl of each dilution was plated on complete minimal medium and on minimal medium lacking adenine. The plates were incubated at 30 °C and photographed after 3–5 days.Kinase Assay—Pulldown and kinase assays were carried out as previously described (5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar), except that the complexes were washed 5 times for 10 min at 4 °C with NETN buffer and once with kinase buffer before carrying out the kinase reaction using 20 μm cold ATP.RESULTSThe ATRMec1/ATMTel1 (S/TQ) Sites of Chk1 Are Required for Its Function in Response to DNA Damage and Replication Blocks—The Chk1 protein consists of two primary domains, the well conserved N-terminal kinase domain and the less conserved non-catalytic C-terminal domain. The current model suggests that in response to DNA damage or replication stress, Chk1 is activated through phosphorylation by ATR/ATM on SQ or TQ motifs on the C-terminal domain. The yeast Chk1 C terminus contains six SQ/TQ sites that could be the phosphorylation targets for the yeast ATR/ATM orthologues, Mec1/Tel1 (Fig. 1). Two of the S/TQ sites, Thr-333 and Thr-382, correspond respectively to the sites, Ser-317 and Ser-345 on mammalian Chk1 known to be phosphorylated in response to DNA damage and replication blocks. Other S/TQ sites in the yeast Chk1 do not seem to be conserved in the mammalian Chk1 proteins.To examine the functional importance of the S/TQ sites of the yeast Chk1, we mutated the CHK1 gene on a plasmid to produce six different chk1 alleles, each conferring alanine substitution of the serine or threonine residue in one of the six S/TQ sites. Although Chk1 is not required for the response to replication blocks by HU in wild-type cells, we have shown that Chk1 is essential in cells that lack Dun1 when grown on HU (7Schollaert K. Poisson J.M. Searle J.S. Scwanekamp J.A. Tomlinson C.R. Sanchez Y. Mol. Biol. Cell. 2004; 15: 4051-4063Crossref PubMed Google Scholar). We, therefore, introduced plasmids carrying wild-type CHK1, mutated chk1, or empty vector into chk1Δ dun1Δ cells to determine whether the mutated Chk1 would rescue the lethality of the chk1Δ dun1Δ cells on HU. chk1Δ dun1Δ cells with an empty vector failed to grow on plates containing 50 mm HU, whereas introduction of a plasmid encoding wild-type CHK1 rescued the lethality (Fig. 2A). The chk1Δ dun1Δ cells harboring plasmids encoding the mutated Chk1T333A failed to grow on HU, whereas cells expressing Chk1T321A, Chk1T356A, and Chk1T382A showed an intermediate phenotype on 50 mm HU and failed to grow on plates containing 80 mm HU. Cells expressing Chk1T376A and Chk1S439A grew as well on HU as cells expressing wild-type Chk1. These results indicated that the three TQ sites at Thr-333, Thr-356, and Thr-382 that gave severe to intermediate phenotypes were important for Chk1 function in response to replication blocks by HU. We, therefore, generated a dun1Δ strain in which the chromosomal CHK1 was mutated to confer alanine substitutions on all the three TQ sites at 333, 356, and 382 (3AQ). As expected, the dun1Δ chk1–3AQ cells, like dun1Δchk1Δ cells, failed to grow on plates containing 50 mm HU (Fig. 2B).FIGURE 2The conserved ATRMec1 phosphorylation sites on Chk1 are essential for the checkpoint response in vivo. A, plasmids carrying Chk1 mutants with alanine substitution of threonine/serine at one of the six putative ATR Mec1 phosphorylation sites were transformed into dun1Δ chk1Δ cells. Transformants were spotted on plates containing 0 or 50 or 80 mm HU. B, the 3AQ mutant combining three point mutations (T333A, T356A, and T382A) was integrated to the CHK1 locus in dun1Δ cells and tested for HU resistance under the same condition. C, chk1Δ cells harboring plasmids carrying HA-tagged Chk1 phosphorylation sites mutants were treated with 0.1% MMS, and protein extracts were analyzed by Western blots using anti-HA antibody. D, left, plasmids carrying HA-tagged 3AQ mutant were introduced into cdc13-1 chk1Δ cells, and DNA damage was elicited by incubating the cells at the nonpermissive temperature. The mobility shifts of Chk1 and accumulation of Pds1 were monitored by Western blots. Right, the 3AQ mutant was integrated into cdc13-1 cells, and checkpoint function of Chk1 in the resulting strains was evaluated using micro-colony assay. The bars represent the average number of cells per colony from 50 colonies. The experiment was carried out in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the phosphorylation of the mutated Chk1 proteins was compromised in response to DNA damage, chk1Δ cells harboring plasmid-borne HA-tagged Chk1 were treated with MMS, and the Chk1 proteins from these cells were analyzed by Western blotting. The wild-type Chk1 from MMS-treated cells exhibited slower migrating forms, indicating modification of the protein in response to DNA damage induced by MMS. Surprisingly, despite the growth phenotypes observed for some of the SQ/TQ mutants on HU, all single AQ mutant proteins exhibited mobility patterns similar to that of wild-type Chk1, although we observed a reduction in the amount of the slowest migrating form in these mutant proteins. However, the triple mutant, Chk13AQ, did not exhibit the slowest migrating forms that were apparent for the wild-type or single mutant proteins (Fig. 2C). These results suggest that phosphorylation or modification of Chk1 took place at multiple sites in addition to the 3TQ sites.DNA damage sensed outside the context of a replication fork can be mimicked in cells by inactivation of Cdc13, a protein that participates in telomere capping (4Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (667) Google Scholar). Chk1 plays a role in the M-A delay after Cdc13 inactivation; thus, we then investigated the role of the 3TQ sites in the DNA damage checkpoint activated by a lesion in late S/G2 induced by a temperature-sensitive allele of CDC13. Inactivation of Cdc13 in cdc13-1 cells at the nonpermissive temperature causes DNA damage that triggers the M-A checkpoint response, which includes stabilization of the securin Pds1 mediated by Chk1 (4Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (667) Google Scholar, 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar, 18Weinert T.A. Hartwell L.H. Genetics. 1993; 134: 63-80Crossref PubMed Google Scholar, 19Cohen-Fix O. Koshland D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14361-14366Crossref PubMed Scopus (141) Google Scholar). A plasmid encoding an HA-tagged Chk13AQ was introduced into a temperature-sensitive strain, cdc13-1 chk1Δ, whose chromosomal PDS1 was also tagged with HA. cdc13-1 cells expressing the Chk13AQ-mutated protein failed to accumulate Pds1 when DNA damage was induced, indicating the Chk13AQ protein was not functional in the checkpoint response (Fig. 2D). Checkpoint activation in cdc13-1 strains results in cell cycle delay at the M-A transition which leads to micro-colonies of 2–4 cells (4Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (667) Google Scholar, 5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar). To determine the contribution of the 3 TQ sites to cell cycle arrest, we generated a cdc13-1 strain in which chk1–3AQ was integrated into its chromosomal locus. We then counted the cells in micro-colonies formed by cdc13-1 cells expressing wild-type CHK1, chk1–3AQv or chk1Δ alleles after triggering the checkpoint via inactivation of Cdc13 at the nonpermissive temperature. As expected, cells expressing wild-type Chk1 formed micro-colonies with only a few cells when grown at the nonpermissive temperature for cdc13-1. In contrast, chk1–3AQ cells had many cells per micro-colony, essentially the same as chk1Δ cells (Fig. 2D). Theses results indicate that the ATRMec1 phosphorylation sites, as defined in the 3AQ mutant, which include Thr-333 and Thr-382 that are conserved in mammalian Chk1, are required for Chk1 function in response to replication blocks and DNA damage sensed in late S/G2.The GD and TKF Domains of Chk1 Mediate Interaction with Rad9 and Are Required for Both the M-A and HU Checkpoint Responses—In addition to the ATRMec1 phosphorylation sites, the C termini of Chk1 proteins have two conserved clusters of amino acids called TRF (TKF in yeast Chk1) and GD domains (11Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1117) Google Scholar) (Fig. 1). To determine the importance of the two domains in Chk1 function, we made two CHK1 deletion mutants, which lacked amino acids (aa) 416–421 (ΔTKF) and aa 496–517 (ΔGD), respectively. dun1Δ and cdc13-1 cells expressing the mutants integrated into the CHK1 loci were examined for their ability to grow on 50 mm HU or to halt the cell cycle in response to DNA damage. The dunΔ chk1-ΔGD cells were unable to grow on 50 mm HU, whereas dun1Δ chk1-ΔTKF cells showed some micro-colonies at higher plating densities after 5 days on HU (Fig. 3A). Both cdc13-1chk1-ΔGD and cdc13-1chk1-ΔTKF failed to elicit the M-A DNA damage checkpoint as shown in the micro-colony assay (Fig. 3C). In addition, both ΔTKF and ΔGD proteins did not generate slower migrating forms when cells were treated with MMS (Fig. 3B), suggesting that the growth and checkpoint phenotypes observed were likely due to absence of Chk1 activation by phosphorylation even though the ATRMec1 sites remained intact in both deletion mutants. We also observed that the Chk1 ΔTKF protein seemed to be unstable as the protein levels of this mutated protein were much lower than that of wild-type protein (Fig. 3B). It is unknown whether the lower protein level contributed to the phenotypes observed for chk1-ΔTKF cells.FIGURE 3The conserved GD and TRF domains in the C terminus of Chk1 are essential for the checkpoint response in vivo. A, the chk1 gene lacking the GD domain (ΔGD) or TKF domain (ΔTKF) was integrated into dun1Δ cells. The resulting strains were evaluated for resistance to replication blocks on 50 mm HU. B, plasmids encoding HA-tagged deletion mutants were introduced into chk1Δ cells. The mobility shifts of Chk1 proteins after MMS treatment were monitored by Western blots. *, cross-reaction signal indicative of the relative amounts of protein loaded. C, the deletion mutants were also integrated into cdc13-1 cells, and their ability to halt the cell cycle was evaluated by micro-colony assay. Numbers for duplicate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The phosphorylation of Chk1 by ATRMec1 is mediated by an adapter/mediator protein, Rad9, which has been shown to physically interact with Chk1 (5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar). It was formally possible that the ΔTKF and ΔGD Chk1 proteins failed to interact with Rad9 and was, thus, incapable of being activated. To test this, we used a two-hybrid system to examine the interaction between Rad9 and wild-type and mutated Chk1 proteins. Rad9 protein fused to the AD of Gal4 and wild-type or mutated Chk1 proteins fused to the DNA binding domain (BD) of Gal4 were co-expressed in yeast cells carrying an ADE2 reporter. Cells expressing AD-Rad9 and BD-Chk1 grew well on plates lacking adenine, whereas cells expressing AD-Rad9 with an empty BD vector or BD-Chk1 with an empty AD vector did not grow (Fig. 4A), confirming the interaction between Chk1 and Rad9. No growth was observed in cells expressing AD-Rad9 with BD-Chk1 proteins lacking the GD or TKF domains (Fig. 4A), indicating that the GD and TKF domains are important determinants for Chk1-Rad9 interaction. However, single amino acid substitutions of invariant residues in the GD (G503) and TKF (F419) domains did not lead to a loss of interaction with Rad9 (Fig. 4A and see “Discussion”). It should be mentioned that the levels of fused BD-Chk1 deletion proteins were comparable to that of the wild-type BD-Chk1 in the cell (data not shown); hence, the lack of interaction between Rad9 and Chk1 deletion mutants was not due to lower levels of Chk1 mutated proteins. Interestingly, although cells expressing Chk1–3AQ fail to grow on HU and fail in the DNA damage checkpoint (Fig. 2), mutation of the 3TQs had no effect on Chk1 interaction with Rad9 (Fig. 4A). Likewise, the catalytically inactive mutant, Chk1-D142A (5Sanchez Y. Bachant J. Wang H. Hu F. Liu D. Tetzlaff M. Elledge S.J. Science. 1999; 286: 1166-1171Crossref PubMed Scopus (452) Google Scholar), also defective in HU response and checkpoint function (data not shown), exhibited normal interaction with Rad9 (Fig. 4A). These results indicate that residues within the GD and TKF domains of Chk1 but not the ATRMec1 phosphorylation sites nor its kinase activity are necessary for the interaction with Rad9. Studies with Chk1 proteins from other organisms and our studies presented here suggest that the Chk1 C terminus acts to inhibit the catalytic activity of the N terminus. Despite this, we did not observe an interaction between the kinase domain and C-terminal domains using this two-hybrid system (Fig. 4B).FIGURE 4The GD and TRF domains but not the ATRMec1 phosphorylation sites on Chk1 are required for interaction with Rad9p. A, two-hybrid reporter cells were co-transformed with a plasmid expressing Rad9 fused to the GAL4 AD and a plasmid carrying wild-type or mutant Chk1 fused to the GAL4 DNA BD. Transformants were spotted in a series of dilutions on the media indicated. Interaction between AD and BD fusion proteins allowed growth on the media lacking adenine. D142A, catalytically inactive mutant. B, the Chk1 N terminus (chk1-NT, 1–304) and the C terminus (chk1-CT, 281–527) were fused to the GAL4 activation and DNA binding" @default.
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