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. 2000 May;11(5):1535-46.
doi: 10.1091/mbc.11.5.1535.

Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends

Z Guo  1 , W G Dunphy
Affiliations
Free PMC article

Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends

Z Guo et al. Mol Biol Cell. 2000 May.
Free PMC article

Abstract

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that Xenopus Cds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-free Xenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast, Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.

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Figures

Figure 1
Figure 1
Sequence of Xcds1. Sequences of Cds1 homologues were aligned with the use of the PrettyPlot function of the Genetics Computer Group (Madison, WI) program. Identical residues are boxed. Sequences that were used to design degenerate PCR primers are underlined. The GenBank accession number for Xcds1 is AF174295.
Figure 2
Figure 2
Modification of Xcds1 in response to M13 DNA and linearized plasmids. (A) Interphase extracts containing 2000 sperm nuclei/μl (lane1), 2000 sperm nuclei/μl and 100 μg/ml aphidicolin (lane 2), or 2000 UV-damaged sperm nuclei/μl (lane 3) were incubated at 23°C for 100 min. Seventy microliters of each extract was centrifuged through a sucrose cushion to isolate the nuclear fractions, which were subjected to SDS-PAGE and immunoblotting. After detection of Xcds1, the immunoblot was stripped and probed with anti-Xchk1 antibodies. (B) Interphase egg extracts containing 10 ng/μl M13 single-stranded (ss) DNA or the same amount of M13 DNA and 5 μg/ml actinomycin D were incubated at 23°C. An aliquot of extract (2 μl) was taken and frozen every 20 min after the addition of M13 DNA. Xcds1 protein in each aliquot was then detected by immunoblotting. (C) Extracts containing various DNA templates (lanes 1–6) or the same DNA templates and 5 mM caffeine (lanes 7–12) were incubated at 23°C for 90 min and then analyzed for Xcds1 protein by immunoblotting. The immunoblot was subsequently stripped and probed for Xchk1 protein.
Figure 3
Figure 3
Modification of Xcds1 in response to various oligonucleotides. (A) Extracts containing DNA homopolymers (lanes 2–5) or the same DNA templates and 5 mM caffeine (lanes 6–9) were incubated at 23°C for 90 min and analyzed for Xcds1 protein by immunoblotting. The immunoblot was stripped and probed for Xchk1 protein. (B) Radiolabeled poly(dT)40 and poly(dC)40 were incubated with 100 μl of interphase extract. Just after the addition of the homopolymers (0 min) and every 15 min (15–90 min) afterward, a 10-μl sample was taken and deproteinized. The homopolymers were detected with autoradiography after native PAGE. (C) Same as A except that the indicated oligonucleotides were used. (D) Interphase egg extracts containing 50 ng/μl oligonucleotide duplex (oligo 1 + oligo 2) were incubated at 23°C. An aliquot of extract (2 μl) was taken and frozen every 10 min after the addition of oligonucleotides. Xcds1 protein in each aliquot was detected by immunoblotting. (E) Xcds1 protein immunoprecipitated from extracts containing 50 ng/μl poly(dT)40 was treated with either λ phosphatase or buffer for 60 min at 30°C. As a control, Xcds1 was also immunoprecipitated from untreated interphase extracts. Xcds1 protein in the immunoprecipitates was examined by immunoblotting. (F) Same as A except that interphase egg cytosol containing 50 ng/μl poly(dT)40 or no DNA was incubated for 100 min.
Figure 4
Figure 4
Activation of Xcds1 kinase in response to poly(dT)40. (A) His6-Xcds1 (WT) and His6-Xcds1-N324A (MUT) were purified from E. coli and incubated with GST-Cdc25[254–316]-WT (WT) or GST-Cdc25[254–316]-S287A (MUT) in kinase buffer containing [32P]ATP. The proteins were separated by SDS-PAGE and stained with Coomassie blue. The phosphorylated proteins were detected with the use of a PhosphorImager. (B) Immunoprecipitation (IP) was performed from interphase egg cytosol containing poly(dT)40 or no DNA with the use of anti-Xcds1 antibodies or nonspecific rabbit IgG. One-third of each immunoprecipitate was analyzed for the modification of Xcds1 by immunoblotting; two-thirds of each immunoprecipitate was incubated with GST-Cdc25[254–316]-WT to measure kinase activity as in A. (C) Quantitation of the data presented in B with the use of a PhosphorImager.
Figure 5
Figure 5
Response of Xenopus egg extracts to poly(dT)40 in the presence and absence of Xcds1. (A) A simple DNA homopolymer delays mitosis. Poly(dT)40 (○, □) or poly(dG)40 (●, ▴) was added to the extracts at a final concentration of 50 ng/μl in the presence (□, ▴) or absence (○, ●) of 5 mM caffeine. The extracts were activated with CaCl2 before the addition of DNA. Sperm nuclei (200 nuclei/μl) were added to the extracts to monitor the timing of nuclear envelope breakdown (NEB) by microscopy. (B) Removal of Xcds1 and Xchk1 proteins from egg extracts by immunodepletion. M-phase extract (100 μl) was incubated with a mixture of anti-Xcds1 antibodies (20 μg) and anti-Xchk1 antibodies (10 μg) bound to Affiprep protein A beads for 50 min at 4°C with constant rocking. Protein A beads were removed by centrifugation. A second round of depletion was then performed to ensure that both Xcds1 and Xchk1 were completely removed, which was assessed by immunoblotting. As a control, M-phase extracts underwent the same procedure with nonspecific rabbit IgG. For depletion of Xcds1 alone, anti-Xchk1 antibodies were omitted. (C) Depletion of Xcds1 does not diminish the mitotic delay caused by poly(dT)40. Sperm nuclei (500 nuclei/μl) (●, ▴) or both sperm nuclei (500 nuclei/μl) and poly(dT)40 (50 ng/μl) (○, □) were added to Xcds1-depleted (○, ●) or mock-depleted (□, ▴) extracts. (D) Same as C except that both Xcds1 and Xchk1 were depleted.
Figure 6
Figure 6
Response of Xenopus egg extracts to an oligonucleotide duplex and M13 DNA in the presence and absence of Xcds1. (A) Depletion of Xcds1 does not diminish the mitotic delay caused by an oligonucleotide duplex. Sperm nuclei (500 nuclei/μl) (●, ▪) or both sperm nuclei (500 nuclei/μl) and oligonucleotide duplex (oligo 1 + oligo 2) (50 ng/μl) (○, □) was added to Xcds1-depleted (○, ●) or mock-depleted (□, ▪) extracts. Sperm nuclei (500 nuclei/μl) were also added to untreated extracts (▴). The timing of nuclear envelope breakdown (NEB) was monitored by microscopy. (B) Same as A except that the response to M13 DNA (10 ng/μl) was examined.
Figure 7
Figure 7
Kinase activity toward Ser-287 of Xenopus Cdc25 in egg extracts lacking Xcds1 and/or Xchk1. (A) M-phase extracts were immunodepleted with anti-Xcds1 antibodies, anti-Xchk1 antibodies, both anti-Xcds1 and anti-Xchk1 antibodies, or nonspecific rabbit IgG. The treated extracts were activated with CaCl2 and incubated at 23°C for 90 min in the presence or absence of poly(dT)40. Finally, total kinase activity toward GST-Cdc25[254–316]-WT (WT) or GST-Cdc25[254–316]-S287A (MU) was assayed in each sample. (B) Quantitation of the data shown in A. Only kinase activity toward GST-Cdc25[254–316]-WT was quantitated, because phosphorylation of the S287A mutant was negligible.
Figure 8
Figure 8
Model for the response of the kinases Xcds1 and Xchk1 to different DNA signals.
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