MCB Accepted Manuscript Posted Online 22 July 2019 Mol. Cell. Biol. doi:10.1128/MCB.00175-19 Copyright © 2019 American Society for Microbiology. All Rights Reserved. 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 4 5 Running Title: A tel2 mutation abolishes replication checkpoint 6 7 Yong-jie Xua,*, Saman Khana, Adam C. Didierb, Michal Wozniaka, Yufeng 8 Liuc, Amanpreet Singha,d, and Toru M. Nakamurab 9 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). 22 23 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. 43 44 45 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). 61 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). 73 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). 95 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.