A Tel2 Mutation That Destabilizes the Tel2-Tti1-Tti2 Complex Eliminates

1 A tel2 mutation that destabilizes the Tel2-Tti1-Tti2 complex eliminates

2 Rad3ATR kinase signaling in the DNA replication checkpoint and leads to

3 telomere shortening in fission yeast

5 Running Title: A tel2 mutation abolishes replication checkpoint

7 Yong-jie Xua,*, Saman Khana, Adam C. Didierb, Michal Wozniaka, Yufeng

8 Liuc, Amanpreet Singha,d, and Toru M. Nakamurab

10 a Department of Pharmacology and Toxicology, Boonshoft School of Medicine,

11 Wright State University, Dayton, Ohio, USA

12 b Department of Biochemistry and Molecular Genetics, University of Illinois at

13 Chicago, Chicago, Illinois, USA

14 c The First Affiliated Hospital of Zhengzhou University, Zhengzhou City, Henan

15 Province, China

16 d Department of Cancer Genetics and Epigenetics, City of Hope Comprehensive 17 Cancer Center, Duarte, California, USA

18 * The corresponding author: [email protected]

19 Key words: Tel2; replication checkpoint; replication stress; hydroxyurea; Rad3;

20 Cds1; Chk1; ATR kinase; chaperone; genome integrity; telomeres.

21 Word counts: Abstract: 191 (limit: 200); Main Text: 7859 (limit: 8000).

1 24 ABSTRACT

25 In response to perturbed DNA replication, ATR (ataxia telangiectasia and

26 Rad3-related) kinase is activated to initiate the checkpoint signaling necessary for

27 maintaining genome integrity and cell survival. To better understand the signaling

28 mechanism, we carried out a large-scale genetic screen in fission yeast looking for

29 mutants with enhanced sensitivity to hydroxyurea. From a collection of ~370

30 primary mutants, we found a few mutants in which Rad3 (ATR ortholog)-

31 mediated phospho-signaling was significantly compromised. One such mutant

32 carried an uncharacterized mutation in tel2, a gene encoding an essential and

33 highly conserved eukaryotic protein. Previous studies in various biological

34 models have shown that Tel2 mainly functions in Tel2-Tti1-Tti2 (TTT) complex

35 that regulates the steady-state levels of all phosphatidylinositol 3-kinase-like

36 protein kinases (PIKKs), including ATR. We show here that although the levels

37 of Rad3 and Rad3-mediated phospho-signaling in DNA damage checkpoint were

38 moderately reduced in the tel2 mutant, the phospho-signaling in DNA replication

39 checkpoint was almost completely eliminated. In addition, the tel2 mutation

40 caused telomere shortening. Since the interactions of Tel2 with Tti1 and Tti2 were

41 significantly weakened by the mutation, destabilization of the TTT complex likely

42 contributes to the observed checkpoint and telomere defects.

2 46 INTRODUCTION

47 DNA replication can be perturbed by various endogenous and exogenous

48 factors. If undetected, perturbed replication forks collapse, causing chromosomal

49 DNA damage or even cell death. To maintain the genome integrity, eukaryotes

50 have evolved a surveillance mechanism called the DNA replication checkpoint

51 (DRC) to monitor fork progression during normal S phase or under stress [see

52 reviews (1,2)]. The DRC senses the problems and activates cellular responses

53 such as increased production of dNTPs, cell cycle delay, fork stabilization, and

54 suppression of late firing origins, which all work in concert to minimize the

55 mutation rate and to ensure accurate duplication of the genome. Consistent with

56 its importance in genome integrity, the DRC is highly conserved from yeasts to

57 humans and defects in the pathway cause a wide range of developmental and

58 cancer-predisposition syndromes. Although debatable, mutations generated by

59 errors in DNA replication followed by mistakes in repair likely contribute

60 significantly to sporadic cancers (3).

62 Studies in the past decades have identified a related set of sensor proteins in

63 all eukaryotes that assemble at perturbed forks for the DRC signaling. Among the

64 sensors, ATR is the kinase that works with its co-factor ATRIP (ATR-interacting

65 protein) and the 9-1-1 (Rad9-Rad1-Hus1) complex to initiate the signaling by

66 phosphorylating various substrates including the checkpoint mediators and

67 effector kinase (4-6). The activated mediators channel the signal to the effector

68 kinase (7,8). Once activated, the effector kinase diffuses away from the fork and

3 69 relays the signal to various cellular structures to stimulate the responses

70 mentioned above. Despite its importance in genome integrity and extensive

71 studies in the past, the DRC signaling mechanism, however, remains incompletely

72 understood (9-11).

74 To better understand the mechanism by which the checkpoint signaling is

75 initiated at perturbed forks (9,10), we have searched for new DRC mutants in the

76 fission yeast Schizosaccharomyces pombe, an established model for studying the

77 cellular mechanisms that are conserved in humans. By random mutation of the

78 genome, a large-scale genetic screen has been carried out to look for mutants with

79 enhanced sensitivity to hydroxyurea (HU). HU has been of clinical and scientific

80 interest for ≥ 100 years (12,13). It perturbs DNA replication by inhibiting

81 ribonucleotide reductase (RNR), a highly conserved enzyme required to provide

82 dNTPs for DNA replication and repair (14). RNR contains a catalytically essential

83 diferric tyrosyl radical inside its smaller subunit. HU quenches the tyrosyl radical

84 and thus suppresses RNR, which slows down polymerase movement at the forks

85 (15,16). Consistent with this mechanism, HU resistance has been observed in cells

86 overexpressing RNR or expressing a mutant RNR (17-19). DNA replication can

87 also be perturbed by DNA damage such as those caused by methyl

88 methanesulfonate (MMS) or ultraviolet (UV) light. Unlike HU, which slows forks

89 globally, DNA damage pauses a subset of on-going forks at the damage sites on

90 the leading strand template (20). In addition, DNA damage, if occurs outside S

91 phase, provokes the DNA damage checkpoint (DDC) responses (Fig. 1A).

4 92 Therefore, HU, if properly used, specifically induces replication stress and studies

93 in various eukaryotic organisms have shown that the primary response to HU

94 treatment is the activation of DRC (2,21,22).

96 Here we report our identification of a previously uncharacterized mutation

97 in tel2 by the hus (HU sensitive) screen that significantly sensitizes S. pombe to

98 HU and the DNA damage agents MMS, UV and bleomycin. Tel2 is an essential

99 and a highly conserved protein among eukaryotes (23,24). It was originally

100 identified in C. elegans and S. cerevisiae ≥ 30 years ago (25-29). The current

101 model suggests that Tel2 functions in the TTT (Tel2-Tti1-Tti2) complex as a co-

102 chaperone that regulates the protein levels of all PIKKs, including ATR, and

103 hence multiple cellular processes (30-33). We found that the level of Rad3ATR was

104 moderately reduced in this mutant similar to that in S. cerevisiae tel2-1 mutant

105 (34,35). Consistent with the reduced Rad3 level, the signaling in the DDC

106 pathway was moderately compromised. Surprisingly, the mutation almost

107 completely eliminated the signaling in the DRC pathway. Interestingly, unlike this

108 S. pombe tel2 mutant and the two C. elegans mutants that are sensitive to

109 replication stress (29), the S. cerevisiae tel2-1 mutant is insensitive to HU and

110 DNA damage (34). Similar to the S. cerevisiae tel2-1 mutant (25), the mutation

111 also caused telomere shortening in S. pombe. Because the mutation significantly

112 weakened the interactions of Tel2 with Tti1 and Tti2, it is likely that the

113 destabilized TTT complex causes the defects in checkpoint signaling and telomere

114 maintenance.

5 115

116 RESULTS

117 Screening of a fission yeast mutant hus227 with enhanced sensitivities to HU

118 and DNA damage. We have carried out a large-scale hus screen in S. pombe

119 looking for new DRC mutants and accumulated ~370 primary mutants. The

120 mutants were backcrossed three times to remove bystander mutations. After

121 removing known mutations by crossing with DRC mutants and other hus mutants,

122 a small set of new hus mutants was screened that likely causes DRC defects. One

123 such mutant is hus227. Preliminary results suggested that phosphorylation of the

124 DRC mediator protein Mrc1Claspin by Rad3 (8,11) was eliminated. We therefore

125 decided to investigate further on hus227.

126

127 We first examined the sensitivities of hus227 to HU and DNA damage by

128 using standard spot assay. Rad3 is the master checkpoint kinase responsible for

129 activation of both DRC and DDC in S. pombe (36) (Fig. 1A). The ATM ortholog

130 Tel1 contributes minimally to checkpoint in fission yeast. Deletion of rad3

131 sensitizes S. pombe to both HU and DNA damage due to a lack of checkpoint

132 functions. As shown in the top panels of Fig. 1B, while S. pombe cells lacking

133 Rad3, Mrc1 or Cds1 were highly sensitive to HU, the cells lacking Chk1, the

134 effector kinase of the DDC, was less sensitive, suggesting that the replication

135 stress induced by HU is mainly dealt by DRC. Under similar conditions, the

136 hus227 mutant was found sensitive to HU and the sensitivity was higher than

137 mrc1 and cds1 but less than rad3. We then examined the sensitivity of hus227 to

6 138 DNA damage caused by MMS and UV (Fig. 1B, middle panels). Unlike the HU

139 treatment, chk1 mutant was more sensitive to MMS and UV than cds1, indicating

140 that the DNA damage that occurs at G2, the major cell cycle stage in S. pombe, is

141 mainly dealt by DDC. The hus227 was also sensitive to MMS and UV and the

142 sensitivity was higher than chk1 but less than rad3. We then examined the

143 sensitivity of hus227 to bleomycin (Fig. 1B, bottom panels), an antibiotic that

144 generates single-strand and double-strand breaks in chromosomal DNA (37).

145 Interestingly, hus227 was highly sensitive to bleomycin and the sensitivity was

146 even higher than rad3 that lacks both DDC and DRC. This suggests that the

147 mutation may also affect DNA repair (see below). We also found that hus227 was

148 unable to grow at 37˚C (Fig. 1C), showing that it is a ts mutant and the mutated

149 gene is likely essential or required for cell growth at 37˚C.

150

151 We have recently screened a new set of hus mutants with remarkable HU

152 sensitivities that are caused by mutations in various metabolic pathways, not DRC

153 (38,39). These mutants are much more sensitive to chronic exposure than to acute

154 HU treatment, and they cannot be rescued by upregulation of Suc22, the small

155 subunit of RNR and the major regulatory target of DRC in fission yeast. We then

156 examined the sensitivity of hus227 to acute HU treatment and found that hus227

157 was sensitive and the sensitivity was comparable to mrc1 but less than rad3

158 mutant (Fig. 1D). We next examined the rescuing effect of Suc22 on hus227.

159 Unlike the metabolic erg11-1 mutant in ergosterol biosynthesis that could not be

160 rescued (38), upregulation of Suc22 rescued hus227 similar to rad3 (Fig. 1E). We

7 161 then performed tetrad dissection on crosses of hus227 with all known DRC

162 mutants as well as S. pombe lacking the nuclear membrane protein Lem2 (40). All

163 crosses generated spores with wild type HU resistance (Fig. S1). This suggests

164 that hus227 is not allelic to any of the tested mutants and likely a new DRC

165 mutant in S. pombe.

166

167 hus227 carries a single missense mutation in tel2. To identify the mutated gene,

168 we transformed hus227 with S. pombe genomic DNA expression libraries

169 carrying an ura4 marker. Colonies grown on plates lacking uracil were replicated

170 onto plates containing HU to screen those with conferred HU resistance. Plasmids

171 recovered from the yeast colonies were subjected to restriction enzyme digestions

172 and subsequent DNA sequencing, which identified tel2. Expression of wild type

173 tel2 on a vector fully rescued hus227 (Fig. 2A). Under the same conditions,

174 mutant tel2 recused to a much lesser degree. Sequencing of the PCR product

175 generated by using a high fidelity polymerase and genomic DNA purified from

176 hus227 identified a single G to A mutation, causing a Cys307 to Tyr amino acid

177 change in Tel2 (Fig. 2B).

178

179 To see whether the mutation is linked to the hus and ts phenotypes of

180 hus227, we integrated wild type and the mutant tel2 tagged with HA epitope at

181 the genomic locus in wild type S. pombe by using the method diagrammed in the

182 top of Fig. 2C. After screening by colony PCRs and backcrosses to ensure single-

183 copy integration in the genome, the integrants was confirmed by PCRs using

8 184 Phusion polymerase (Fig. 2C, lower panel) and Western blotting with anti-HA

185 antibody (Fig. S2). We then examined the HU sensitivities of the integrants and

186 found that while the integrant of wild type tel2 was resistant to HU, the integrant

187 of mutant tel2 was sensitive (Fig. 2D). The mutant integrant not only showed the

188 HU sensitivity similar to hus227, but the integration also caused a similar ts

189 phenotype (Fig 2D, top right panel). We also assessed the HU sensitivity at 25˚C

190 and found that both hus227 and the mutant integrant were sensitive, although to a

191 lesser degree (Fig. 2D, lower panels). This suggests that the mutation uncouples

192 the essential role of Tel2 in cell growth and the function in HU resistance. We

193 then quantified the protein levels of Tel2 by Western analysis using Ponceau S

194 staining as the loading control because it is linear with protein concentration (R2 =

195 0.99) (41) (Fig. 2E). The results showed that when cultured at 30˚C, the level of

196 endogenous mutant Tel2 was reduced to 80.2% (± 4.1, n=4) of wild type level. At

197 25 ˚C, the level of mutant Tel2 was similar to that in wild type cells (Fig. S2 and

198 S7). When expressed on a vector, both wild type and mutant Tel2 were increased

199 by > 2 folds, which may explain the partial rescuing effect of mutant Tel2 shown

200 in Fig. 2A. Together, we found that both hus and ts phenotypes of hus227 were

201 caused by the single mutation in tel2. We hereafter renamed hus227 as tel2-

202 C307Y. Throughout the rest of this study, all experiments were carried out in

203 YE6S medium at 30˚C except when use of different temperatures is specified.

204

205 Tel2 was first identified in S. cerevisiae (24,25) and C. elegans (26-28,42)

206 with a wide range of defects in genome integrity, maintenance of telomeres,

9 207 lifespan, and development. Previous studies in various models have shown that

208 Tel2 mainly functions, together with Hsp90 and several co-factors, in regulating

209 protein stability of all PIKKs and hence the multiple biological processes (30-33).

210 Although Tel2 is conserved in eukaryotes, similarity in overall amino acid

211 sequences is limited (Fig. S3). Similar to the mutated residues Ser129 in S.

212 cerevisiae tel2-1 and Cys135 and Cys772 in C. elegans rad-5 and clk-2, respectively,

213 the mutated Cys307 residue in tel2-C307Y is not highly conserved. It was therefore

214 not a surprise that expression of human Tel2 failed to rescue tel2-C307Y (Fig. S4).

215 Considering that the mutation may perturb metabolism via other PIKKs and thus

216 sensitizes S. pombe to HU, we examined the HU sensitivity of tel2-C307Y cells

217 containing various auxotrophic markers. The result showed that auxotrophies

218 contributed minimally to HU sensitivity (Fig. S5). By measuring the bulk

219 phosphorylation of Mrc1 in a tel2 shut-off strain, an earlier study suggests that

220 Tel2 may function in DRC in S. pombe (43). However, depletion of Tel2

221 minimally sensitized S. pombe to HU and UV and, more importantly, the cut or

222 “cell untimely torn” cells, which occur in all HU-treated DRC mutants, were

223 rarely observed, which raises a concern of the observed DRC defect (43). In

224 addition, the DRC defect was not examined in details due to a lack of phospho-

225 specific antibodies. Since our newly screened tel2-C307Y mutant is highly

226 sensitive to HU and DNA damage, we decided to further investigate this mutant

227 and its potential defect in DRC by using the set of phospho-specific antibodies

228 that we have made available in the previous studies (11,44).

229

10 230 HU arrests tel2-C307Y cells in S phase. As mentioned above, HU arrests some

231 of the metabolic hus mutants such as erg11-1 and hem13-1 at G2/M, not S phase,

232 as they are killed by HU via a mechanism unrelated to replication stress (38,39).

233 We first examined the cell cycle progression of tel2-C307Y by flow cytometry

234 (Fig. 3A). When incubated with 15 mM HU for 1 to 3 h, wild type cells were

235 increasingly arrested at S phase. Further incubation did not completely stop DNA

236 synthesis as the cells managed to continue the S phase progression and eventually

237 finished the bulk of DNA synthesis in ~7 or 8 h. rad3 and mrc1 cells were also

238 arrested in S phase after 3 h HU treatment. However, these checkpoint mutants,

239 and rad3 in particular, could not properly synthesize DNA in HU. Under similar

240 conditions, HU arrested majority of tel2-C307Y at S phase although a small

241 number of cells remained at G2/M after 3 to 4 h incubation. Unlike wild type cells,

242 however, tel2-C307Y failed to continue DNA synthesis in HU similar to rad3 and

243 mrc1. This result strongly suggests a DRC defect in tel2-C307Y.

244

245 The DRC defect in tel2-C307Y. In the presence of HU, the DRC is activated to

246 delay mitosis so that the cells have adequate time to complete DNA synthesis

247 before they divide. The DRC mutants, however, proceed into mitosis, generating

248 the cut phenotype in which two daughters do not have equal amounts or lack

249 detectable genomic DNAs. We stained the cells with Hoechst for genomic DNAs

250 and Blankophor for septum and examined cell septation during the course of HU

251 treatment (Fig. 3B). After wild type cells were treated with 15 mM HU for 3 h,

252 cell division was almost completely suppressed and remained suppressed during

11 253 the rest of HU treatment, suggesting an activated DRC. In contrast, rad3 mutant

254 showed a robust cell division activity in HU. >50% of cells underwent cell

255 division after 3 h HU treatment and continued to divide during the rest of the

256 treatment. As a result, most of the cells were short and showed the cut phenotype

257 (Fig. 3C, arrows). The mrc1 mutant initially slowed down cell division during the

258 first 1 to 3 h. After 3 to 4 h in HU, cells began to divide, generating the cut cells

259 (Fig. 3C). However, unlike the HU-treated rad3 cells that were short due to the

260 lack of both DRC and DDC, the mrc1 cells elongated because the collapsed forks

261 activates DDC (8,40). Similar to rad3 and mrc1 mutants and consistent with the

262 cell-killing effect shown in Fig. 1D, tel2-C307Y cells continued to divide in the

263 presence of HU and the septation index was higher than mrc1 but lower than rad3

264 (Fig. 3B). Furthermore, since the HU-treated tel2-C307Y cells were shorter than

265 mrc1 but longer than rad3, the mutation may also affect the DDC (see below).

266

267 Next, we examined cell recovery from HU (Fig. 3D). After 4 h treatment

268 with 15 mM HU, almost all wild type, rad3, and mrc1 cells and majority of tel2-

269 C307Y cells were arrested at S phase. When released in fresh medium, wild type

270 cells could fully recover and returned to normal cell cycle in ≤ 4 h. In contrast,

271 rad3 and mrc1 cells could not finish DNA synthesis and failed to recover.

272 Similarly, tel2-C307Y also failed to recover from the HU arrest. Together, we

273 show that the tel2-C307Y mutation causes a profound DRC defect, which

274 sensitizes S. pombe to the replication stress induced by HU or DNA damage.

275

12 276 Elimination of Rad3 phospho-signaling in DRC. Under replication stress, Rad3

277 phosphorylates two functionally redundant residues Thr645 and Thr653 in Mrc1 and

278 Thr412 in Rad9 of the 9-1-1 complex (Fig. 1A, left). Phosphorylation of Mrc1 and

279 Rad9 facilitates the phosphorylation of Thr11 in Cds1CHK2/Rad53 by Rad3, which

280 promotes the autophosphorylation of Cds1-Thr328 in the activation loop.

281 Phosphorylation of Thr328 directly activates Cds1, leading to full activation of the

282 DRC in S. pombe (8,11,44). When DNA damage occurs outside S phase or forks

283 collapse causing strand breaks, such as the situations in HU-treated mrc1 or cds1

284 cells, Rad3 phosphorylates Rad9 and Crb253BP1/Rad9 (45-47), which in turn

285 facilitate the phosphorylation of Chk1-Ser345 by Rad3 (48,49), leading to the

286 activation of DDC (Fig. 1A, right).

287

288 To investigate the DRC defect in tel2-C307Y, we examined Rad3-mediated

289 phosphorylation of Rad9, Mrc1 and Cds1 by using phospho-specific antibodies

290 (8,11,44). Under physiological conditions, Rad9 is phosphorylated at a basal level

291 mainly by Rad3 in wild type cells (compare wild type cells with rad3, tel1, and

292 rad3 tel1 mutants in Fig. 4A). After the cells were treated with 15 mM HU for 3 h,

293 phosphorylation of Rad9 was significantly increased. In tel2-C307Y mutant, the

294 basal Rad9 phosphorylation remained detectable under normal conditions. After

295 HU treatment, however, the phosphorylation was not increased similar to that in

296 HU-treated rad3 cells. Next, we examined Rad3-specific phosphorylation of

297 Mrc1-Thr645, a representative of the two redundant sites in Mrc1 (Fig. 4B) (8).

298 After HU treatment, Mrc1 phosphorylation was significantly increased in wild

13 299 type cells. Under similar conditions, Mrc1 phosphorylation was reduced to an

300 undetectable level in tel2-C307Y. Similar to rad3, the protein level of Mrc1 in

301 HU-treated tel2-C307Y was lower than in wild type cells, which is consistent with

302 the lack of DRC because DRC upregulates Mrc1 (50). We also examined Mrc1

303 phosphorylation after the cells were treated with 0.01% MMS for 90 min. Similar

304 results were observed (Fig. 4C). Finally, we examined Rad3-dependent

305 phosphorylation of Cds1 and found that the phosphorylation was almost

306 completely eliminated in HU (Fig. 4D) or MMS (Fig. 4E). Together, these results

307 show clearly that the tel2-C307Y mutation almost completely abolished Rad3

308 kinase signaling in DRC.

309

310 Reduced Rad3 phospho-signaling in DDC. Next, we examined the Rad3-

311 dependent phosphorylation of Rad9 and Chk1 in the DDC pathway (Fig. 1A,

312 right). Similar to the HU treatment (Fig. 4A), treatment with 0.01% MMS for 90

313 min significantly increased Rad9 phosphorylation by Rad3 in wild type cells (Fig.

314 5A). However, Rad9 phosphorylation was not readily increased in MMS-treated

315 tel2-C307Y cells. We then examined the phosphorylation of Chk1-Ser345 using the

316 phospho-specific antibody developed by the Walworth lab (48) (Fig. 5B). To our

317 surprise, unlike Cds1 phosphorylation shown in Fig. 4 D and E, ≥50% of Chk1

318 phosphorylation remained detectable in MMS-treated tel2-C307Y cells. To

319 confirm this result, we examined Chk1 phosphorylation in the presence of

320 increasing concentrations of MMS (Fig. 5C). Quantitation results clearly showed

321 that although reduced, majority of the Chk1 phosphorylation remained in tel2-

14 322 C307Y (Fig. 5D). Since Tel2 functions in multiple cellular processes, it is possible

323 that other kinases may participate in Chk1 phosphorylation in tel2-C307Y. We

324 then examined the phosphorylation in tel2-C307Y lacking either Rad3 or Tel1

325 (Fig. 5E). The result showed clearly that the major kinase responsible for Chk1

326 phosphorylation in tel2-C307Y was Rad3, which excluded this possibility.

327 Together, these results show that although Rad3 signaling was almost completely

328 eliminated in DRC, it remained partially functional in DDC. This result is

329 consistent with the observation in Fig. 3C in which the HU-treated tel2-C307Y

330 cells were slightly longer than rad3.

331

332 Moderate reduction of Rad3 and Tel1 in tel2-C307Y. Studies in multiple

333 organisms have shown that Tel2 regulates the stability of all PIKKs, including

334 ATR and ATM (30,33-35,51). We next measured the levels of Rad3 and Tel1 in S.

335 pombe. Although not vigorously examined, the numbers of Rad3 and Tel1

336 molecules in a S. pombe cell are much lower than in S. cerevisiae and mammalian

337 cells (52). We tagged Rad3 N-terminus with myc epitope at its genomic locus.

338 The tagging does not affect the function of Rad3 as the tagged strain showed a

339 wild type HU resistance (Fig. 6A). To ensure accurate quantification, 3 separate

340 samples were analysed by using Ponceau S staining as the loading controls (41).

341 We found that Rad3 in tel2-C307Y was reduced to 60.1% (± 1.3, n = 3) of wild

342 type level (Fig. S6). To see whether the kinase activity of Rad3 contributes to its

343 instability in tel2-C307Y, we examined the kinase inactive rad3-D2249E (36) and

344 found that the kinase activity was unrelated with the instability (Fig. 6C). Since

15 345 Rad3 complexes with Rad26ATRIP/Ddc2, we also examined Rad26 and found that in

346 contrast to Rad3, the level of Rad26 was significantly increased by >2 folds in

347 tel2-C307Y (Fig. 6D and S6). This shows that Tel2 specifically regulates the

348 stability of Rad3, not its cofactor Rad26, which is different from human cells in

349 which depletion of Tel2 decreases the levels of both ATR and ATRIP (53).

350

351 We then tagged and examined Tel1, the ATM ortholog in S. pombe.

352 Because our preliminary data suggested that the number of Tel1 per cell was even

353 lower than Rad3 and could not be reliably detected by Western blotting in whole

354 cell extracts, we examined Tel1 after enrichment by IP. Multiple samples of equal

355 number of cells were collected, lysed, and IPed for Western blotting (Fig. 6E).

356 The results showed that similar to Rad3, Tel1 was reduced to 38% (± 33.7, n = 5)

357 of its wild type level in tel2-C307Y (Fig. S6). Together, these data showed that

358 both Rad3 and Tel1 were moderately reduced in tel2-C307Y. Whether the

359 mutation affects the protein stability of other PIKKs, although likely, remains to

360 be investigated. Interestingly, the double mutant containing tel2-C307Y and

361 kinase inactive rad3 grew significantly slower than the single mutants under

362 normal conditions (Fig. 6A). This indicates that the remaining Rad3 in tel2-

363 C307Y is active that plays an important role in promoting the cell growth (see

364 below).

365

366 Shortened telomeres in tel2-C307Y. Since Rad3 and Tel1 play redundant but

367 essential roles in recruitment of telomerase to telomeres in S. pombe (54,55), we

16 368 expected that tel2-C307Y might carry shorter telomeres. In S. cerevisiae, Tel2

369 directly binds to telomeric DNA and regulates telomere length (25,56). In the two

370 C. elegans tel2 mutants, reports showed clear changes or no change in telomere

371 length (29,42,57). Overexpression of Tel2 in human cells gradually lengthens

372 telomeres (58). Although acute depletion of Tel2 in S. pombe does not alter

373 telomere length (43), the tel2-C307Y mutation may make an accumulative effect

374 on telomere through generations. To investigate, we examined telomere length in

375 tel2-C307Y after restreaking on plates for 5 times at either 25˚C or 32˚C. Genomic

376 DNAs were prepared, digested with EcoRI, and processed for Southern blotting

377 (Fig. 6F). We found that tel2-C307Y cells showed significant telomere shortening

378 for both temperatures. Quantification results from three independent experiments

379 showed that at 25˚C, the average telomere length in tel2-C307Y was 290bp (n=5),

380 which was significantly shorter than that in wild type cells (385bp, n=6) (Fig. 6G).

381 At 32˚C, the length further shortened to 156bp (n=6) in tel2-C307Y as compared

382 with the wild type length of 317bp (n=6).

383

384 Consistent with the previous in vitro data of S. cerevisiae Tel2 (56) that

385 found direct binding of Tel2 to telomeric DNA, ChIP (chromatin

386 immunoprecipitation) assays detected weak telomere association of Tel2 that is

387 eliminated in tel2-C307Y (Fig. 6H). In addition, we found that Rad26 associated

388 with telomeres and the association was also significantly reduced to almost the

389 background level in tel2-C307Y, suggesting that association of Rad3-Rad26 with

390 telomeres is dependent on Tel2. Strong reduction in Rad26 binding in tel2-C307Y

17 391 is especially remarkable as Rad26 protein level is actually increased in this mutant

392 (Fig. 6D and S6). Taken together, these results clearly demonstrate that Tel2 is

393 required to maintain wild-type telomere length in S. pombe by modulating Rad3-

394 Rad26 action at telomeres.

395

396 Destabilization of the TTT complex. Tel2 binds to Tti1 and Tti2 to form the

397 TTT complex, which interacts directly with newly synthesized PIKKs and

398 functions as a co-chaperone for proper folding of these large protein kinases

399 (30,31). Since the levels of Rad3 and Tel1 were reduced in tel2-C307Y, we then

400 investigated whether the mutation affects the interactions of Tel2 with Tti1 and

401 Tti2 by co-IP (co-immunoprecipitation). Tti1 and Tti2 were tagged with myc

402 epitope at their genomic loci following the similar strategy used for tel2

403 integration (Fig. 2C). The tagged strains were crossed into S. pombe expressing

404 endogenously HA tagged wild type or mutant Tel2. The co-IP experiments were

405 carried out multiple times and representative results are shown in Fig. 7A-E. We

406 found that all three proteins could be successfully IPed. When Tti1 and Tti2 were

407 IPed (Fig. 7A and C), Tel2 was co-IPed. When Tti1 and Tti2 were IPed from tel2-

408 C307Y cells, much less Tel2 was co-IPed, suggesting weaker interactions. The

409 intensities of the co-IPed Tel2 bands were quantified and normalized with the

410 inputs (Table S4, sheet 7). After removal of non-specific bindings, we found that

411 the average levels of co-IPed Tel2 were reduced to 14.2% (n=3) and 26.5% (n=4)

412 of wild type levels for Tti1 and Tti2, respectively (Fig. 7E). In the reciprocal co-

413 IPs, the mutation reduced the levels of co-IPed Tti1 (Fig. 7B) and Tti2 (Fig. 7D)

18 414 to 26.6% (n=5) and 15.1% (n=4) of wild type levels, respectively (Fig. 7E and

415 sheet 7 in Table S4). Clearly, the TTT complex was significantly destabilized by

416 the tel2-C307Y mutation.

417

418 Other potential functions of Tel2 in S. pombe. Since Rad3 kinase activity

419 promotes normal growth of tel2-C307Y (Fig. 6A), we then crossed tel2-C307Y

420 with other checkpoint mutants and examined cell growth of the double mutants

421 under normal conditions or in the presence of MMS, UV or HU (Fig. 8A). The

422 results showed that among all double mutants tested, rad3∆ tel2-C307Y cells had

423 the most severe growth defect, suggesting that Rad3 might play additional role

424 other than checkpoint activation that is especially important in promoting the cell

425 growth of tel2-C307Y. Conversely, since the double mutant of rad3∆ tel2-C307Y

426 and all other tested double mutants were more sensitive to MMS, UV and HU,

427 Tel2 may have other functions that are genetically separable from the checkpoint

428 function. Interestingly, the double mutants containing crb2 or chk1 in DDC were

429 significantly more sensitive to MMS and UV than the double mutants containing

430 mrc1 and cds1 in the DRC pathway. Because HU specifically induces S phase

431 stress and MMS and UV mainly cause DNA damage at G2, this result is

432 consistent with the observed major defect of tel2-C307Y in DRC, not DDC (Fig. 4

433 and 5). Furthermore, with a compromised DDC, DNA damage caused by MMS

434 and UV may be carried over to the next cell cycle (59), exacerbating the

435 replication stress and thus enhancing the lethality. Since DNA strand breaks that

436 occur at G2 are less likely to be carried over to next cell cycle (60,61), we then

19 437 examined the sensitivity of chk1∆ tel2-C307Y double mutant to bleomycin (Fig.

438 8B) with the expectation that this mutant might show less synthetic lethality as

439 compared with that in MMS or UV treatment. Surprisingly, similar to the MMS

440 and UV treatments, the chk1∆ tel2-C307Y mutant as well as the double mutants

441 containing rad3 or cds1 was significantly more sensitive to bleomycin than the

442 single mutants. It is possible that with a compromised DDC, DNA strand breaks,

443 particularly single strand breaks, can also be carried over to next cell cycle. The

444 reduced Tel1 level in the double mutants may also explain the higher sensitivities

445 because Tel1 can activate Chk1 under certain specific conditions (62,63). As

446 mentioned above, because tel2-C307Y was more sensitive to bleomycin than rad3,

447 Tel2 may also regulate DNA strand break repair or other undefined cellular

448 processes.

449

450 Because tel2-C307Y is a ts mutant, we examined the cell cycle progression

451 after the cultures were shifted to 37˚C. At 25˚C, the mutant behaved almost like

452 wild type cells (Fig. S7A). After culturing at 37˚C, Tel2 gradually decreased to a

453 minimally detectable level in ~12 h (Fig. S7B). Meanwhile, DNA contents began

454 to increase and a fraction of the mutant cells showed 8C to 16C DNA contents

455 after 4 to 6 h, which was minimally detected in wild type cells (Fig. S7A). This

456 suggests DNA re-replication or misregulation of cell division. We then examined

457 the cells under microscope after staining with Hoechst and Blankophor (Fig. S7C).

458 Most wild type cells contained a condensed nucleus before or after they were

459 incubated at 37˚C for 12 h. In contrast, the nuclei in some mutant cells were less

20 460 condensed or relaxed after culturing at 37˚C. Since cells with multiple septa were

461 never observed, acute depletion of Tel2 may cause DNA re-replication although

462 other possibilities remain. Consistent with this result, a previous study showed

463 that S. pombe lacking tel2 had a defect in S phase entry (43). Together, these

464 results suggest a potential role of Tel2 in DNA replication. Further studies are

465 needed to investigate this possibility.

466

467 DISCUSSION

468 Our genetic screen has identified a previously uncharacterized tel2-C307Y

469 mutation that significantly sensitizes S. pombe to HU and DNA damage. At

470 cellular level, the mutation causes cut cells in HU and the number of cut cells

471 corroborates the cell-killing effect of HU in this mutant. At molecular level, the

472 mutation almost completely eliminates the Rad3 kinase signaling in DRC, which

473 explains the cut phenotype as well as the hypersensitivities of the mutant to all

474 tested replication stresses. We also measured the protein level of Rad3 and found

475 it was moderately reduced. Although this moderate reduction of Rad3 may

476 explain the partial signaling defect in DDC, it remains unclear why DRC is

477 preferentially affected. Nonetheless, although the budding yeast tel2-1 mutant

478 shows an almost wild type resistance to replication stress (34), the DRC defect in

479 S. pombe tel2-C307Y mutant is likely conserved in higher eukaryotes as enhanced

480 sensitivity to replication stress has also been described in the two C. elegans tel2

481 mutants (29) and in mammalian cells (53,64) in which Tel2 is depleted.

482

21 483 Tel2 interacts with Tti1 and Tti2 to form the TTT complex, which interacts

484 with R2TP (Rvb1-Rvb2-Tah1-Pih1) complex (65,66) to bring Hsp90 to PIKKs

485 for their maturation. Interruption of the interactions between Tel2 and its client

486 kinases reduces their steady-state levels, leading to a wide range of phenotypes

487 including the checkpoint defect. Consistent with this model, we found that

488 although the levels of Rad3 and Tel1 were moderately reduced in tel2-C307Y, the

489 mutation significantly weakened the interactions of Tel2 with Tti1 and Tti2. Thus,

490 the reduced Rad3 level is likely caused by destabilization of the TTT complex.

491 The remaining Rad3 in tel2-C307Y is clearly functional because Rad3-dependent

492 Chk1 phosphorylation was still detectable. Furthermore, the kinase activity of

493 Rad3 promotes cell growth of tel2-C307Y and its co-factor Rad26 is significantly

494 increased in the mutant, which likely compensates the partial loss of Rad3. The

495 reduced DDC signaling may leave some DNA lesions “unnoticed” at G2 that are

496 likely carried over to the next cell cycle and thus aggravate the replication stress.

497 Therefore, the hypersensitivity of tel2-C307Y to DNA damage is likely caused by

498 the lack of DRC exacerbated by a compromised DDC. It is also possible that the

499 mutation affects other functions of Tel2 such as DNA repair, leading to the

500 hypersensitivity to DNA damage, particularly those caused by bleomycin.

501

502 The most striking observation in this study is that although a reasonable

503 amount of functional Rad3 remains in tel2-C307Y, its signaling in DRC is almost

504 completely abolished. The hypersensitivity of the mutant to HU and DNA damage

505 suggests that Tel2 may function at or close to the checkpoint sensors. The

22 506 decreased Rad3 level is clearly related to the checkpoint defect. However, why a

507 moderate reduction of Rad3 has such a great impact on DRC, not DDC remains

508 unclear. Here, we propose several hypotheses that need further investigations:

509 first, in S. cerevisiae, checkpoint is more tolerant to DNA damage or damage-like

510 structures during S phase (67). A similar threshold mechanism may also exist in S.

511 pombe. When Rad3 level is reduced, the threshold is raised to a level where the

512 DRC completely “ignores” the replication stress. Second, as a co-chaperone, Tel2

513 may help to assemble DRC proteins to the forks. For example, the highly charged

514 and disordered Mrc1 is specifically expressed during G1/S transition (8) and a

515 non-essential component of replisome (68-70). The tel2-C307Y mutation may

516 affect the assembly of Mrc1 and/or other checkpoint proteins and hence the DRC

517 defect. Consistent with this possibility, overexpression or acute depletion of Tel2

518 in mammalian cells causes checkpoint defects and increased sensitivity to

519 replication stress even before ATR level is reduced (53,58,64). Third, the data

520 shown in Fig. S7 and a previous study in S. pombe lacking tel2 (43) suggest that

521 Tel2 may regulate DNA replication. Defects in this process may increase the

522 checkpoint threshold in S phase (67) mentioned above and explain the slower S

523 phase arrest of tel2-C307Y cells in HU (Fig. 3A and D). Finally, as described

524 above, Tel2 may function in DNA repair, which is known to influence checkpoint

525 signaling.

526

527 Tel2 physically interacts with telomeric DNA and regulates telomere length

528 in S. cerevisiae (25,56). Although it remains controversial whether Tel2 functions

23 529 in the maintenance of telomere length in C. elegans, overexpression of Tel2 in

530 human cells gradually lengthens telomeres (58). Our results show that similar to

531 that in S. cerevisiae, Tel2 associates with telomeres and functions in the

532 homeostasis of telomere length in S. pombe. Furthermore, Tel2 was found to be

533 important for recruitment of Rad26 to telomeres. While Rad3 and Tel1 do play

534 redundant roles in phosphorylating telomere protein Ccq1 (54,55), Rad3-Rad26

535 complex is the primary kinase that phosphorylates Ccq1 to maintain normal

536 telomere length. Thus, rad3∆ and rad26∆ cells show significantly shorter but

537 stable telomeres due to the residual action of Tel1. On the other hand, tel1∆ cells

538 do not show any telomere shortening, and association of Tel1 at telomere cannot

539 be detected in S. pombe cells (71). Thus, short but stable telomeres observed in

540 tel2-C307Y, combined with significant loss of Rad26 association at telomeres,

541 suggest that Tel2's role in telomere length regulation is primarily through

542 regulation of the Rad3-Rad26 kinase complex, although we cannot entirely rule

543 out the possibility that Tel2 might also affect Tel1 function at telomeres.

544

545 MATERIALS AND METHODS

546 Yeast strains and plasmids. The S. pombe strains were usually cultured at 30°C

547 in YE6S (0.5% yeast extract, 3% dextrose and 6 supplements) or in EMM

548 medium lacking the appropriate supplements following standard methods (72).

549 Yeast strains, plasmids, and PCR primers used in this study are listed in

550 Supplementary Table S1, S2, and S3, respectively. All cloned genes and the

551 mutations were confirmed by DNA sequencing (Retrogen).

24 552

553 The hus screen. Screening for new hus mutants was carried out as previously

554 described (38,39). Briefly, wild type S. pombe was mutagenized with

555 methylnitronitrosoguanidine and saved at 4˚C (73). ~3000 – 5000 mutagenized

556 cells were spread on each YE6S plate. The colonies were replicated onto YE6S

557 plates containing 5 mM HU to screen those that were sensitive to HU (Sigma).

558 The primary hus mutants were crossed with known hus mutants to identify novel

559 mutants and then backcrossed three times to remove bystander mutations.

560

561 Integration of the tel2-C307Y mutation. The tel2-C307Y expression cassette

562 was cloned into the pBS cloning vector between BamHI and MluI sites. An HA

563 tag linked with an ura4 marker were inserted in frame at the 3’ end of tel2 (see

564 diagram in Fig 2C). After digestion with PstI and XhoI, the 6470bp integration

565 fragment was gel purified and then transformed into wild type TK7 strain.

566 Colonies formed on plates lacking uracil were replicated onto HU plates.

567 Integrants that were sensitive to HU were screened by colony PCRs for both 5’

568 and 3’ ends. The screened integrants were also backcrossed once to ensure single

569 copy integration in the genome. Genomic DNAs were then purified from the

570 integrants for PCRs using primers SpTel2(P)f and SpTel2(T)MluI-b and Phusion

571 polymerase (NEB) to confirm the integration at the tel2 locus. Western blotting

572 with anti-HA antibody confirmed the proper tagging and integration.

573

574 Drug sensitivity. Sensitivities to HU and various DNA damaging agents were

25 575 determined by standard spot assay or in liquid medium as described in our

576 previous studies (8,11,38,39). Briefly, for the spot assay, 2 x 107 cells/ml of

577 logarithmically growing S. pombe were diluted in five-fold steps and spotted in 3

578 µl onto YE6S plates or YE6S plates containing the drugs at indicated

579 concentrations. The YE6S plates spotted with the cells were dried before the

580 treatment with UV (Stratalinker 2400). The plates were incubated at 30°C for 3

581 days or 25˚C for 4 days and then photographed. All spot assay experiments were

582 repeated at least once. To examine the sensitivity to acute HU treatment (73), HU

583 was added to liquid YE6S medium at 15 mM. At each time point, an equal

584 number of cells were removed, diluted 1000-fold, spread onto three YE6S plates,

585 and incubated at 30°C for 3 days for cell recovery. Colonies were counted and

586 presented as percentages of the untreated cells.

587

588 IP and co-IP. 1 x 108 logarithmically growing cells were harvested and saved at -

589 20˚C in a 1.5 ml screw cap tube. The frozen cell pellets were lysed by mini-bead

590 beater in the buffer containing 25 mM HEPES/NaOH (pH7.5), 50 mM NaF, 1

591 mM NaVO4, 10 mM NaP2O7, 40 mM ß-glycerophophate, 0.1% Tween 20, 0.5%

592 NP-40, and protease inhibitors. The lysates were centrifuged at 16,000 g, 4˚C for

593 5 min to make the cell extract. Anti-HA or anti-myc antibody agarose resins

594 (Santa Cruz) were washed with Tris-buffered saline containing 0.05% Tween 20

595 (TBS-T) three times and incubated with 5% BSA in TBS-T for ≥ 30 min at 4˚C.

596 The cell extract was incubated with the prewashed antibody resins by rotating in 2

597 ml tubes at 4˚C for 2 h. The resins were washed with TBS-T at 4˚C for 20 min,

26 598 repeated three times. The IPed samples were separated by SDS-PAGE followed

599 by Western blottings with anti-HA or anti-myc antibodies.

600

601 Western blotting. The phospho-specific antibodies against phosphorylated Mrc1-

602 Thr645, Rad9-Thr412 and Cds1-Thr11 and their specificities were described in our

603 previous studies (8,11,44). The phospho-specific antibody against phosphorylated

604 Chk1-Ser345 was kindly provided by Dr. Walworth (48). Western analyses of

605 phosphorylated Rad9-Thr412, Mrc1-Thr645 and Cds1-Thr11 have been described in

606 our previous study (11). Rad3, Tel1, Rad26, Tel2, Tti1 and Tti2 were tagged with

607 myc or HA epitope at their genomic loci and examined by Western blotting by

608 using mouse monoclonal antibodies against the myc (9E10, Thermo Scientific) or

609 HA (12CA5, Sigma). For the Western analyses, 1 x 108 logarithmically growing

610 cells were fixed in 15% trichloroacetic acid (TCA) on ice for ≥ 3h and then lysed

611 by mini-bead beater. The lysates from 2 - 4 x 106 cells were separated by SDS-

612 PAGE. Rad3, Rad26 and Tel2 were directly detected in whole cell lysates by

613 Western blotting using Ponceau S staining as the loading control. Tel1 was IPed

614 before the Western analysis. The blotting signal was detected by

615 electrochemiluminescence using ChemiDoc XRS Imaging system (BioRad).

616 Intensities of the specific bands were quantified and analysed by ImageLab

617 (BioRad).

618

27 619 Flow cytometry. 1 x 107 logarithmically growing cells were collected, fixed in

620 ice-cold 70% ethanol, and then analysed by Accuri C6 flow cytometer as

621 described in our previous studies (38,39).

622

623 Microscopy. The cells were fixed directly onto uncoated glass slides by heating

624 briefly at 75˚C for 30 sec or in medium containing 2.5% glutaraldehyde at 4˚C for

625 ≥ 3 h. The glutaraldehyde-fixed cells were washed with PBS by centrifugation at

626 2300 g for 30 sec, stained in the same buffer with 5 µg/ml Hoechst33258 (Sigma-

627 Aldrich) and 1:100 dilution of the Blankophor working solution (MP

628 Biochemicals). The stained cells were examined using an Olympus EX41

629 fluorescent microscope. Images were captured with an IQCAM camera (Fast1394)

630 using Qcapture Pro 6.0 software. ~150 cells were counted for each sample and

631 repeated three times. Images were also extracted into Photoshop (Adobe) to

632 generate Fig. 3C and Fig. S7C.

633

634 ChIP assay.

635 Logarithmically growing cells were grown at 30˚C and processed for ChIP assay

636 using monoclonal anti-myc or anti-HA antibodies, and analyzed by quantitative

637 PCR as prviously described (74). Primers used for ChIP assays (75) are listed in

638 Table S3.

639

640 Telomere length analysis by Southern blot. Wild type and tel2-C307Y cells

641 were restreaked ≥ 5x on YES plates (>120 generations) at either 25˚C or 32˚C.

28 642 Logarithmically growing cells at either temperature were collected to prepare

643 genomic DNAs, which were digested with EcoRI, and then processed for

644 Southern blot analyses with telomere probe as previously described (54). The gels

645 of three independent experiments were quantified with ImageQuant software (Fig.

646 S8) for calculating the telomere length (Table S4, sheet4). Since EcoRI site is

647 ~750bp away from telomere repeat track in fission yeast, 750bp is subtracted to

648 estimate the length of telomere repeats.

649

650 ACKNOWLEDGEMENT

651 We thank Drs. Tom Kelly and Paul Russell for sharing the yeast strains, Dr.

652 Walworth for Chk1 phospho-specific antibody, and Dr. Michael Kemp and three

653 anonymous reviewers for critical reading of the manuscript. Other members of the

654 Xu lab are acknowledged for their help and support. This work was supported by

655 NIH RO1 grants GM110132 to Y.J.X. and GM078253 to T.M.N.

656

657 ABBREVATIONS: ATM: ataxia telangiectasia mutated; ATR: ataxia

658 telangiectasia and Rad3 related; BLM: bleomycin; cut: cell untimely torn; ChIP:

659 Chromatin Immunoprecipitation; DDC: DNA damage checkpoint; DRC: DNA

660 replication checkpoint; HU: hydroxyurea; hus: hydroxyurea sensitive; IP:

661 immunoprecipitation; MMS: methylmethanesulfonate; RNR: ribonucleotide

662 reductase; PIKKs: phosphatidylinositol 3-kinase-like protein kinases; TCA:

663 trichloroacetic acid; UV: ultraviolet.

29 664

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887

39 888

889

890 FIGURE LEGENDS

891 FIG 1. Sensitivity of the newly identified hus227 mutant to HU and DNA

892 damage. (A) Schematics of Rad3 kinase signaling in the DRC (left) and the DDC

893 (right) pathways of S. pombe. Rad3-specific phosphorylation sites are indicated.

894 When DNA replication is perturbed, Rad3 phosphorylates Mrc1 and Cds1 to

895 activate the DRC (8,11,44). When DNA damage occurs outside S phase or forks

896 collapse, such as that in HU-treated mrc1 or cds1 cells, Rad3 phosphorylates

897 Crb253BP1/Rad9 and Chk1 to stimulate the DDC responses (46,48,49).

898 Phosphorylation of Rad9 in the 9-1-1 complex is required for activation of both

899 DRC and DDC (11,45). While phosphorylated Rad9 recruits Rad4TopBP1/Dpb11 to

900 promote Crb2 and Chk1 activation, it remains unclear how phosphorylated Rad9

901 promotes Cds1 activation (dash line). (B) Sensitivities of hus227 mutant (YJ1496)

902 to HU and MMS and UV and bleomycin (BLM) were examined by standard spot

903 assay. Logarithmically growing cells were diluted in five-fold steps and spotted

904 onto YE6S plates as the control and for UV treatment or YE6S plates containing

905 the indicated drugs. The plates were incubated at 30˚C for 3 days and then

906 photographed. Wild type (TK48) and rad3 (NR1826), mrc1 (YJ15), cds1

907 (GBY191) and chk1 (TK197) checkpoint mutants were used as controls. The

908 experiment was repeated three times and a representative result is shown. (C) The

909 ts phenotype of hus227 was examined by spot assay. (D) Sensitivity of hus227 to

910 acute HU treatment. The strains used in B were incubated in YE6S medium

40 911 containing 15 mM HU. At the indicated time points, cells were spread onto YE6S

912 plates to recover for 3 days. Colonies were counted and presented in percentages.

913 Error bars represent means and SEMs of triplicates (Table S4, sheet 1). (E) Over-

914 expression of the RNR small subunit Suc22 rescued hus227. Suc22 was expressed

915 in S. pombe on a vector under the control of its own promoter. The metabolic

916 erg11-1 mutant was used as a negative control (38). V: vector alone.

917

918 FIG 2. The hus and ts phenotypes of hus227 are caused by a single mutation

919 in tel2. (A) Wild type and mutant tel2 was expressed in the indicated strains on a

920 vector under its own promoter. HU sensitivity was examined by standard spot

921 assay. (B) DNA sequencing of tel2 in hus227 identified a G to A mutation that

922 converts Cys307 to Tyr in Tel2. (C) Strategy for integrating the tel2 mutation at its

923 genomic locus (upper panel). HA and nmtT represent HA epitope and nmt1

924 terminator, respectively. Integrants were screened by colony PCRs (red lines) and

925 backcrossed to ensure single copy integration in the genome. Genomic DNAs

926 were purified from the integrants (YJ1513 and YJ1515) and wild type cells (TK48)

927 for PCRs to confirm the integration at tel2 locus (green line). The PCR products

928 were analysed by agarose gel electrophoresis (lower panel). (D) HU sensitivities

929 of wild type (YJ1513) and mutant tel2 integrants (YJ1515) were determined by

930 standard spot assay at 30˚C (upper panels) or 25˚C (lower panels). The same

931 strains were also spotted on YE6S plates and incubated at 37˚C for 2 days to

932 examine the ts phenotype (upper right panel). (E) Expressing on a vector

933 increased the protein levels of Tel2. Wild type (YJ1513) and mutant (YJ1515)

41 934 tel2-HA integrants carrying a vector or the vector expressing wild type or mutant

935 tel2-HA were lysed by TCA method and analysed by Western blotting. A section

936 of Ponceau S stained membrane was used as the loading control. Intensities of the

937 Tel2 bands were quantified and shown in percentages on the bottom.

938

939 FIG 3. The DRC defect in tel2-C307Y. (A) Cell cycle analysis of tel2-C307Y.

940 Wild type, rad3, mrc1 and tel2-C307Y cells used in Fig. 1B were incubated with

941 15 mM HU and analysed by flow cytometry every hour during the incubation.

942 Red lines indicate 1C and 2C DNA contents. (B) tel2-C307Y undergoes cell

943 division in the presence of HU. The cells were incubated with HU as in A and

944 fixed in 2.5% glutaraldehyde at each time point. The fixed cells were stained with

945 Hoechst and Blankophor for microscopic examination. ≥150 cells were counted

946 for each sample, repeated 3x (Table S4, sheet 2). The cells with a septum are

947 shown in percentages. (C) tel2-C307Y cells showed the cut phenotype in HU.

948 Cells were treated with 15 mM HU for 6 h as in A, fixed onto glass slides by brief

949 heating, and stained as in B for microscopic examination. Arrows indicate the cut

950 cells. (D) tel2-C307Y cells failed to recover from HU arrest. The strains used in A

951 were treated with 15 mM HU for 4 h and then released in fresh medium. Cell

952 cycle progression was monitored every hour during the release.

953

954 FIG 4. Elimination of Rad3 kinase signaling in the DRC pathway. (A) HU-

955 induced phosphorylation of Rad9 by Rad3 was eliminated in tel2-C307Y. Wild

956 type and the mutant S. pombe cells were treated with (+) or without (-) 15 mM

42 957 HU for 3 h. Rad9-HA was IPed and separated by SDS-PAGE for Western blotting

958 with anti-HA antibody (lower panel). The same blot was stripped and reprobed

959 with the phospho-specific antibody (upper panel). The Rad9 phosphorylation

960 bands were quantified, and the band intensities in comparison to HU-treated wild

961 type cells are shown at the bottom. (B) HU-induced phosphorylation of Mrc1 by

962 Rad3 was reduced to an undetectable level in tel2-C307Y. Wild type and mutant

963 cells used in Fig. 1B were treated with 15 mM HU for 3 h. Phosphorylation of

964 Mrc1 (upper) was detected with phospho-specific antibodies in whole cell lysates

965 prepared by the TCA method. The same blot was stripped and reprobed with anti-

966 Mrc1 antibodies (mid panel). A section of Ponceau S-stained membrane is shown

967 (bottom panel). (C) MMS-induced phosphorylation of Mrc1 was also reduced to

968 an undetectable level in tel2-C307Y. The cells were treated with 0.01% MMS for

969 90 min and then lysed for Western blotting as in B. (D) HU-induced

970 phosphorylation of Cds1 by Rad3 was eliminated in tel2-C307Y. Wild type, rad3

971 and tel2-C307Y cells were treated with (+) or without (-) HU. Cds1-HA was IPed

972 and then analysed by Western blotting with anti-HA antibody (bottom panel). The

973 same membrane was stripped and re-probed with the phosphor-specific antibody

974 (upper two panels). The Cds1 phosphorylation bands were quantified and shown

975 at the bottom. (E) MMS-induced phosphorylation of Cds1 was also eliminated in

976 tel2-C307Y. The cells were treated with 0.01% MMS for 90 min and analysed as

977 in D.

978

43 979 FIG 5. Moderate reduction of Rad3 kinase signaling in DDC. (A) MMS-

980 induced phosphorylation of Rad9 by Rad3 was significantly reduced in tel2-

981 C307Y. The cells were treated with 0.01% MMS for 90 min, analysed and

982 quantified as in Fig. 4A. (B) MMS-induced phosphorylation of Chk1 by Rad3

983 was only moderately reduced in tel2-C307Y. Wild type, rad3 and tel2-C307Y

984 cells were treated with MMS as in A. Chk1-HA was IPed from an equal number

985 of cells for Western blotting with anti-HA antibody (lower panel). The same blot

986 was stripped and reprobed with the phospho-specific antibody (48) for

987 phosphorylated Chk1 (upper panel). The Chk1 phosphorylation bands were

988 quantified and shown at the bottom. (C) Chk1 phosphorylation was examined and

989 quantified as in B after the cells were treated with increasing concentrations of

990 MMS for 90 min. (D) Intensities of the Chk1 phosphorylation bands in C were

991 quantified and shown in percentages. (E) MMS-induced phosphorylation of Chk1

992 in tel2-C307Y was dependent on Rad3. Chk1 phosphorylation was examined and

993 quantified as in B in wild type, tel2-C307Y, or tel2-C307Y cells lacking rad3 or

994 tel1.

995

996 FIG 6. Despite showing only moderate reduction in Rad3 and Tel1 protein

997 levels, tel2-C307Y cells show telomere length maintenance defect. (A)

998 Inactivation of Rad3 caused synergistic growth defect in tel2-C307Y. Wild type

999 (YJ1138) and kinase inactive (kd) rad3-D2249E (YJ1139) were tagged with myc

1000 epitope at its genomic locus. After crossing with wild type and tel2-C307Y strains,

1001 colonies were selected for examining cell growth under normal conditions or in

44 1002 the presence of HU by standard spot assay. (B) Western analysis of Rad3 levels in

1003 colonies containing wild type or mutant tel2. Cell lysates were prepared by TCA

1004 method for Western blotting. Rad3 was detected by anti-myc antibody, quantified,

1005 and shown in percentages on the bottom and Fig. S6. Asterisk indicates a cross-

1006 reactive material. Ponceau S staining was used as the loading control. (C)

1007 Reduction of Rad3 level in tel2-C307Y was unrelated to the kinase activity of

1008 Rad3. rad3-D2249E was tagged, crossed into tel2-C307Y cells and analysed as in

1009 B. (D) Rad26 level increased in tel2-C307Y. Rad26 was similarly tagged and

1010 analysed as Rad3 in B. (E) Tel1 was tagged with myc epitope at the genomic

1011 locus in wild type (LS8284) or tel2-C307Y mutant (YJ1541). Equal number of the

1012 cells was collected in triplicates, lysed, and IPed. Untagged strain was used as the

1013 control. Relative intensities of Tel1 bands are shown on the bottom and in Fig. S6.

1014 (F) Shortened telomeres in tel2-C307Y mutant. Genomic DNAs were purified

1015 from wild type (TN3783 and TN3784) and tel2-C307Y (YJ1495 and YJ1496)

1016 cells, digested with EcoRI, and processed for Southern blotting with a telomere

1017 probe (54). A representative gel from three independent experiments is shown. (G)

1018 The Southern blots were quantified with ImageQuant (Fig. S8). The telomere

1019 lengths in the indicated strains were calculated by comparing with the standard

1020 markers (Table S4, sheet 4). Means and SEMs in each column are derived from 5

1021 or 6 samples. *** p< 0.001. (H) ChIP analysis to monitor telomere association of

1022 Rad26 and Tel2. Real-time PCR was used to quantify IPed telomeric DNA

1023 relative to input DNA samples. Means and SEMs are calculated from 7 and 5

45 1024 independent experiments for Rad26 and Tel2, respectively (Table S4, sheets 5 and

1025 6). ** p < 0.01; *** p< 0.001.

1026

1027 FIG 7. Weakened interactions of Tel2-C307Y with Tti1 and Tti2. (A) Co-IP of

1028 Tel2 with Tti1 was significantly compromised by tel2-C307Y mutation. Tti1 was

1029 myc tagged at the genomic locus (SK6), crossed into S. pombe containing HA

1030 tagged wild type (SK7) or mutant tel2 (SK9). Tti1 was IPed (upper panel) using

1031 anti-myc antibody to detect co-IPed Tel2 (lower panel) as described in Materials

1032 and Methods. Untagged Tti1 strain (YJ1513) was used as the negative control. 2.4%

1033 cells extracts were loaded as inputs (left three lanes). A section of the Ponceau S-

1034 stained inputs is shown. Tel2 specifically co-IPed with Tti1 was quantified,

1035 normalized with the inputs, and shown underneath the Tel2 blot. A representative

1036 of 5 independent co-IPs is shown. (B) Reduced amount of Tti1 co-IPed with Tel2

1037 in tel2-C307Y. Tel2 was IPed in the same strains as in A with anti-HA antibody to

1038 detect the co-IPed Tti1. (C) Similar as in A, Tti2 was myc tagged in wild type

1039 (YJ1549) and tel2-C307Y (YJ1550) cells. Tel2 co-IPed with Tti2 in wild type and

1040 tel2-C307Y cells was quantified, normalized and shown under the Tel2 blot. A

1041 representative of 3 independent co-IPs is shown. (D) Reciprocal co-IP of Tti2

1042 with Tel2 was carried out in wild type and tel2-C307Y cells similar to B. (E) The

1043 co-IPed bands in A, B, C and D were quantified and normalized with the inputs.

1044 After removing the non-specific bindings, the intensities of co-IP bands from tel2-

1045 C307Y cells are shown in percentages (brown columns) relative to that from wild

1046 type cells (light blue columns). Means and SEMs are derived from 5 and 3

46 1047 independent experiments for Tti1-Tel2 and Tti2-Tel2 co-IPs, respectively (Table

1048 S4, sheet 7).

1049

1050 FIG 8. Increased drug sensitivities of the double mutants containing tel2-

1051 C307Y and checkpoint mutations. (A) Sensitivities of the single and double

1052 mutants containing tel2-C307Y and the indicated checkpoint mutations to MMS,

1053 UV and HU were examined by standard spot assay. Dash lines indicate

1054 discontinuity. (B) Sensitivities of the indicated single and double mutants

1055 containing tel2-C307Y and rad3, cds1 in DRC and chk1 in DDC to bleomycin.

1056