Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism

<div><p>The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in <i>DDC2</i>, called <i>ddc2-S4</i>. The <i>ddc2-S4</i> mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The <i>ddc2-S4</i> mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.</p></div

Regulation of protein kinase Mec1 after DNA damage.

<p>(A) Mec1 activation after MMS treatment. Cells expressing Mec1-HA (KSC1635) or Mec1-KN-HA (KSC1645) were cultured at 25°C and incubated with nocodazole to arrest at G2/M. Cells were then either mock treated (−) or treated (+) with 0.05% MMS for 1 hr. Immunoprecipitated Mec1-HA was subjected to the <i>in vitro</i> kinase assay using GST-Rad53, a recombinant protein purified from <i>E. coli</i>, as a substrate. Incorporation of ³²P into GST-Rad53 was monitored by phosphorimager. The amounts of Mec1 or Mec1-KN in the immunoprecipitates were determined by immunoblotting with anti-HA antibody. Phosphorylation of GST-Rad53 was normalized to that observed with immunoprecipitated mock treated wild-type Mec1. Relative phosphorylation is determined from three independent experiments. (B) Mec1 activation kinase after incubation with MMS for various lengths of time. Cells were treated with MMS for indicated lengths of time and were subjected to the <i>in vitro</i> kinase assay as in A. (C) Effect of <i>ddc1Δ dpb11-1</i> mutation on Mec1 activation. Wild-type (KSC1333) and <i>ddc1Δ dpb11-1</i> (KSC3130) cells expressing Mec1-HA protein were subjected to the <i>in vitro</i> kinase assay as in A. (D) Effect of <i>ddc2Δ</i> mutation on Mec1 activation. Wild-type (KSC1635) and <i>ddc2Δ</i> (KSC1636) cells expressing HA-Mec1 protein were subjected to the <i>in vitro</i> kinase assay as in A.</p

List of oligonucleotides used in this study.

<p>List of oligonucleotides used in this study.</p

Effect of <i>ddc2-S4</i> on Mec1-Ddc2 interaction and Mec1 localization.

<p>(A) Effect of <i>ddc2-S4</i> mutation on Mec1-Ddc2 interaction. <i>MEC1-HA ddc2Δ</i> (KSC1340) or <i>ddc2Δ</i> (KSC1234) cells were transformed with YCp-DDC2-myc or YCp-DDC2-S4-myc. Extracts prepared from cells were subjected to immunoprecipitation with anti-HA antibodies. Immunoprecipitates and whole extracts were analyzed by immunoblotting with anti-HA or anti-myc antibodies. (B) Effect of <i>ddc2-S4</i> or <i>ddc2-KA</i> mutation on Mec1 localization to an HO-induced DSB. Wild-type (KSC1635), <i>ddc2-S4</i> (KSC2158) or <i>ddc2-KA</i> (KSC2159) cells expressing Mec1-HA were transformed with the YCpA-GAL-HO plasmid. Transformed cells were grown in sucrose and treated with nocodazole. After arrest at G2/M, the culture was incubated with galactose for 3 hr to induce HO expression, while half of the culture was maintained in sucrose to repress HO expression. (Top) The strains contain an HO cleavage site, marked with <i>HIS2</i>, at the <i>ADH4</i> locus on chromosome (Chr.) VII. The HO1 primer pair amplifies a region 1 kb away from the HO cleavage site. An arrow represents the telomere. (Bottom) Cells were subjected to chromatin immunoprecipitation with anti-HA antibodies. Association of Mec1 with an HO-induced DSB was analyzed by real-time PCR. Relative enrichment was determined from three independent experiments. (C) Effect of <i>ddc2-S4</i> on Rad53 phosphorylation after co-localization of Ddc1-LacI and Ddc2-LacI to a LacO array. Cells containing the LacO₂₅₆ array and <i>RAD53-HA</i> with the combination of DDC1-LacI-GFP and DDC2-LacI-GFP (CBY88) or DDC2-S4-LacI-GFP (KSC2419) under the control of GAL promoter were grown in sucrose and arrested in nocodazole for 2 hr. Galactose was pulsed for 2 hr, and then cells were further incubated with glucose. Samples were collected at 2 hr after addition of glucose and subjected to immunoblotting analysis with anti-HA or anti-GFP antibodies.</p

Identification of the Ddc2 region required for Mec1 interaction and DNA damage response using the modified two-hybrid assay.

<p>The two-hybrid tester <i>ddc2Δ</i> strain (KSC2077) carrying pBD-MEC1(2-2368) was transformed with pAD-DDC2, pAD-DDC2-ΔC, pAD-DDC2-ΔN1, pAD-DDC2-ΔN2, pAD-DDC2-ΔN3 or pAD-DDC2-S4. The coiled-coil domain (amino acid 68–138) is indicated as a gray bar and the KKRK sequence (amino acid 177–180) is shown as KR. The <i>ddc2-S4</i> mutation carries two substitution mutations (K263E and H382Y). Transformants were streaked on an SD-Ura-Leu-His plate containing 1 mM AT, YEPD plate containing 1 mg/ml HU or 0.005% MMS. Interaction with Mec1 and DNA damage sensitivity were assessed by cell proliferation on the respective plates.</p

DNA damage response and proliferation of <i>ddc2-S4</i> mutants.

<p>(A, B) DNA damage sensitivity of <i>ddc2-S4</i> mutants. Serial dilutions of cultures were spotted on YEPD medium without or with MMS or HU (A). Cultures spotted on YEPD medium were irradiated with UV light (B). Plates were incubated at 30°C for two-three days. Strains used were wild-type (KSC1178), <i>ddc2Δ</i> (KSC1234) and <i>ddc2-S4</i> (KSC3153). (C) Effect of <i>ddc2-S4</i> on DNA damage checkpoint signaling. Wild-type (KSC1178), <i>ddc2Δ</i> (KSC1234), and <i>ddc2-S4</i> (KSC3153) cells expressing Rad53-HA were grown to log-phase and exposed to MMS (0.05%) at 30°C for the indicated length of time. Cells were harvested and subjected to immunoblotting analysis with anti-HA antibodies. (D) G2/M-phase DNA damage checkpoint in <i>ddc2-S4</i> mutants. Wild-type (KSC1178), <i>ddc2Δ</i> (KSC1234) or <i>ddc2-S4</i> (KSC3153) cells were arrested with nocodazole and irradiated or not irradiated with UV (50 J/m²). At the indicated times after release of UV-irradiated (+UV) and unirradiated (−UV) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining. (E) Effect of <i>ddc2-S4</i> mutation on cell proliferation in the presence of <i>SML1</i>. <i>ddc2Δ</i> mutants carrying the <i>URA3</i>-marked YCp-DDC2 plasmid (KSC3308) were transformed with YCpT-DDC2, YCpT-DDC2-S4 or the control vector. Transformants were streaked and grown on plates containing medium with or without 5-fluoroorotic acid (5-FOA) at 30°C. Only cells that have lost the <i>URA3</i>-marker plasmid can proliferate in the presence of 5-FOA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004136#pgen.1004136-Boeke1" target="_blank">[62]</a>.</p

Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl <i>cis–trans</i> Isomerase from <i>Escherichia coli</i>

FKBP22, a protein expressed by <i>Escherichia coli</i>, possesses PPIase (peptidyl-prolyl <i>cis</i>-<i>trans</i> isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with <i>Legionella</i> Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding–unfolding mechanism of Mip-like proteins, we investigated a recombinant <i>E. coli</i> FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD⁺ (NTD with the entire flexible region), and CTD⁺ (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD⁺ and CTD⁺ seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD⁺

Chymotrypsin and trypsin digested His-CI fragments were analyzed by Tris-Tricine SDS-16

<p><b>Copyright information:</b></p><p>Taken from "Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity"</p><p>http://www.virologyj.com/content/4/1/64</p><p>Virology Journal 2007;4():64-64.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934351.</p><p></p>5% PAGE followed by silver staining. Molecular masses (in kDa) of marker proteins are shown at the left side of gel. 'Chy' and 'Try' indicate chymotrypsin and trypsin, respectively whereas, a – h indicate intact repressor, different digested fragments of repressor, respectively. N-terminal ends of fragments c and h were sequenced. Western blotting analysis of chymotrypsin/trypsin digested His-CI fragments from 2 and 30 mins incubations by a standard procedure as indicated in Materials and method. Summary of proteolysis. The putative domains of CI and its amino acid residues involved in formation of hinge, NTD and CTD are indicated

Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity-2

<p><b>Copyright information:</b></p><p>Taken from "Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity"</p><p>http://www.virologyj.com/content/4/1/64</p><p>Virology Journal 2007;4():64-64.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934351.</p><p></p>as calibrated with BSA (66 kDa, I), ovalbumin (46 kDa, II), carbonic anhydrase (29 kDa, III), and lysozyme (14.4 kDa, IV). Molecular weights were plotted against /, where and denote elution volume and void volume respectively. Void volume of column was determined from elution of blue dextran. Glutaraldehyde (GCHO) cross-linking. Nearly 0.5 μM His-CI or CTD was cross-linked with 0.1% GCHO and samples were analyzed by SDS-10% PAGE. Protein bands were visualized by silver staining. Horizontal arrows denote dimeric His-CI and CTD species