細胞分裂週期因子7 (Cdc7)是一個參與DNA複製過程中很重要的、從酵母菌到人類都有的絲氨酸/蘇氨酸激酶。當它和Dbf4或Drf1調節因子結合後就會成為具有活性的激酶。當細胞進入複製期時， Cdc7-Dbf4激酶參與DNA複製起始、穩定複製叉和S週期檢查點。 我們先前的研究結果顯示當Cdc7在癌症細胞中過度表現時，會加快雙股斷裂的產生而活化檢查點訊號，而且還會增加同源重組DNA修復的能力。然而, Cdc7如何調節同源重組DNA修復和促使腫瘤生成的詳細機制依然未知。Mre11-Rad50-Nbs1 (MRN) 複合體是同源重組DNA修復過程之關鍵起始步驟中不可或缺的分子。它們會在雙股斷裂的終端上產生3’端的單股DNA，即所謂核苷酸修剪。它是一種在DNA損傷反應,可吸引許多DNA修復的蛋白質形成複合體。我們發現，Cdc7-Dbf4激酶會和Mre11有交互作用。更有趣地, 在DNA損傷時, Mre11的磷酸化與Cdc7和ATR激酶的活性有關。然而, 根據in vitro kinase assay的實驗結果, Cdc7並不會直接磷酸化Mre11，而是間接地調控Mre11的磷酸化。接著發現, 抑制Cdc7活性會抑制ATR磷酸化下游受質如RPA2及Nbs1的能力，並抑制DNA修復的蛋白質結合到毀損的染色質DNA上。 總結， 這些結果顯示在紫外線壓力下, Cdc7-Dbf4透過ATR調節Mre11鄰酸化來調節同源重組DNA修復。這項研究暗示過量表現的Cdc7-Dbf4在複製壓力下調控檢查點活化和促進同源重組DNA修復進而促使腫瘤生成，對於癌症治療提供了一個新的方向。

The cell division cycle 7 (Cdc7) is a serine-threonine kinase, conserved from yeast to human, whose activation requires the interaction with either one of the regulatory subunits dumb-bell forming factor 4 (Dbf4) or diaphanous-related formin 1 (Drf1). Cdc7-Dbf4 kinase participates in the initiation of DNA replication, replication fork stability, and the S-phase checkpoint upon replication stress. Our recent findings suggest that overexpression of Cdc7 accumulates double-stranded breaks (DSBs) to activate checkpoint signaling and enhances the capacity of homologous recombination (HR) repair. However, the detailed mechanism of how up-regulated Cdc7 promotes HR repair and tumorigenesis remains obscure. The critical control of the HR takes place at the initiation step, which entails nucleolytic resection of the DSB ends to generate ssDNA with 3’-termini. This resection process needs Mre11–Rad50–NBS1 (MRN) complex that has multiple roles in the DNA damage response (DDR) including direct detection of DSB and amplification of damage signal. In this study, we showed here that Cdc7-Dbf4 kinase interacts with Mre11. Interestingly, Mre11 phosphorylation upon DNA damage is dependent on the kinase activity of Cdc7 and ATR. However, Mre11 phosphorylation is indirectly regulated by Cdc7 according to in vitro kinase assay. We then found that inhibition of Cdc7 activity impairs the phosphorylation of RPA2 and Nbs1 by ATR and the recruitment of HR repair proteins such as Rad51 on chromatin in response to replication stress. Collectively, these results suggest a novel function of Cdc7-Dbf4 that regulates HR repair through ATR-mediated Mre11 phosphorylation following UV stress. This study sheds new light on the tumorigenic role of Cdc7-Dbf4 in regulating checkpoint activation and HR repair upon replication stress, which provides a new direction to cancer therapy combination.

誌謝詞…………………………………………………………………………………….….Ⅰ
中文摘要……………………………………………………………………………….…..Ⅱ
Abstract……………………………………………………………………………………...Ⅲ
Abbreviations……………………………………………………………………………..Ⅳ
Chapter 1. Literature Review and Research Aims…………………...…..1
1.1 Cancer…………………………………………………………………………………………………….1
1.2 DNA Replication……………………………………………………………………………………..1
1.3 DNA Damage Checkpoint and DNA Repair……………………………………………..2
1.4 Homologous recombination DNA repair…………………………………………………5
1.5 Mre11-Rad50-Nbs1 Complex………………………………………………………………...5
1.6 Research Aims…………………………………………………………………………………………7
Chapter 2. Materials and Methods………………………………………………9
2.1 Cell culture……………………………………………………………………………………………..9
2.2 Drug treatment……………………………………………………………………………………….9
2.3 Transfection…………………………………………………………………………………………..10
2.4 Whole cell lysate preparation………………………………………………………………..10
2.5 Immunoblotting…………………………………………………………………………………….10
2.6 Immunopreciptation……………………………………………………………………………..11
2.7 In vitro kinase assay……………………………………………………………………………….11
2.8 Antibodies………………………………………………………………………………………………12
2.9 Chromatin isolation……………………………………………………………………………….12
2.10 Plasmid Construction……………………………………………………………………………12
2.11 Ligation of Mre11 fragments into pET-28a (+)-modified
expression vector………………………………………………………………………………….13
2.12 Agarose Gel Electrophoresis of DNA…………………………………………………….13
2.13 shRNA infection……………………………………………………………………………………13
2.14 Adenovirus Construction and infection……………………………………………….14
Chapter 3. Results……………………………………………………………………….15
3.1 Overexpression or knockdown of Cdc7 regulate Mre11
phosphorylation level…………………………………………………………………….15
3.2 Cdc7 and Dbf4 is able to interact with Mre11…………………………………16
3.3 Cdc7 is involved in Mre11 phosphorylation not through
a direct phosphorylation…………………………………………………………….….16

3.4 ATM/ATR regulates the phosphorylate of Mre11…………………………….17
3.5 Cdc7 is important to Mre11 phosphorylation and ATR-
mediated checkpoint activity and HR repair……………………………….….18
Chapter 4. Discussion…………………………………………………………………...20
Figures…………………………………………………………………………....23
Reference…………………………………………………………………….….41

Aguilera, A. and B. Gomez-Gonzalez (2008). "Genome instability: a mechanistic view of its causes and consequences." Nature reviews. Genetics 9(3): 204-217.
Bakkenist, C. J. and M. B. Kastan (2003). "DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation." Nature 421(6922): 499-506.
Bartek, J., C. Lukas, et al. (2004). "Checking on DNA damage in S phase." Nature reviews. Molecular cell biology 5(10): 792-804.
Bartek, J., C. Lukas, et al. (2004). "Checking on DNA damage in S phase." Nat Rev Mol Cell Biol 5(10): 792-804.
Bell, S. P. and A. Dutta (2002). "DNA replication in eukaryotic cells." Annual review of biochemistry 71: 333-374.
Bousset, K. and J. F. Diffley (1998). "The Cdc7 protein kinase is required for origin firing during S phase." Genes & development 12(4): 480-490.
Carney, J. P., R. S. Maser, et al. (1998). "The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response." Cell 93(3): 477-486.
Cerosaletti, K. M., A. Desai-Mehta, et al. (2000). "Retroviral expression of the NBS1 gene in cultured Nijmegen breakage syndrome cells restores normal radiation sensitivity and nuclear focus formation." Mutagenesis 15(3): 281-286.
Chapman, J. R. and S. P. Jackson (2008). "Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage." EMBO Rep 9(8): 795-801.
Cheng, L., T. Collyer, et al. (1999). "Cell cycle regulation of DNA replication initiator factor Dbf4p." Molecular and cellular biology 19(6): 4270-4278.
Cho, S. H., C. D. Toouli, et al. (2005). "Chk1 is essential for tumor cell viability following activation of the replication checkpoint." Cell Cycle 4(1): 131-139.
Cho, W. H., Y. J. Lee, et al. (2006). "CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein." Proceedings of the National Academy of Sciences of the United States of America 103(31): 11521-11526.
Cimprich, K. A. and D. Cortez (2008). "ATR: an essential regulator of genome integrity." Nat Rev Mol Cell Biol 9(8): 616-627.
Cimprich, K. A. and D. Cortez (2008). "ATR: an essential regulator of genome integrity." Nature reviews. Molecular cell biology 9(8): 616-627.
Di Virgilio, M., C. Y. Ying, et al. (2009). "PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin." DNA repair 8(11): 1311-1320.
Di Virgilio, M., C. Y. Ying, et al. (2009). "PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin." DNA Repair (Amst) 8(11): 1311-1320.
Donaldson, A. D., W. L. Fangman, et al. (1998). "Cdc7 is required throughout the yeast S phase to activate replication origins." Genes & development 12(4): 491-501.
Dong, Z., Q. Zhong, et al. (1999). "The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage." J Biol Chem 274(28): 19513-19516.
Duncker, B. P. and G. W. Brown (2003). "Cdc7 kinases (DDKs) and checkpoint responses: lessons from two yeasts." Mutat Res 532(1-2): 21-27.
Falck, E., M. Patra, et al. (2005). "Response to comment by Almeida et al.: free area theories for lipid bilayers--predictive or not?" Biophysical journal 89(1): 745-752.
Falck, J., J. Coates, et al. (2005). "Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage." Nature 434(7033): 605-611.
Gao, Q., X. Huang, et al. (2006). "Influence of chk1 and plk1 silencing on radiation- or cisplatin-induced cytotoxicity in human malignant cells." Apoptosis 11(10): 1789-1800.
Golub, E. I., R. C. Gupta, et al. (1998). "Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA." Nucleic Acids Res 26(23): 5388-5393.
Harper, J. W. and S. J. Elledge (2007). "The DNA damage response: ten years after." Molecular cell 28(5): 739-745.
He, T. C., S. Zhou, et al. (1998). "A simplified system for generating recombinant adenoviruses." Proc Natl Acad Sci U S A 95(5): 2509-2514.
Iftode, C., Y. Daniely, et al. (1999). "Replication protein A (RPA): the eukaryotic SSB." Crit Rev Biochem Mol Biol 34(3): 141-180.
Ilves, I., T. Petojevic, et al. (2010). "Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins." Molecular cell 37(2): 247-258.
Jackson, A. L., P. M. Pahl, et al. (1993). "Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein." Molecular and cellular biology 13(5): 2899-2908.
Jares, P., A. Donaldson, et al. (2000). "The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?" EMBO Rep 1(4): 319-322.
Jares, P., A. Donaldson, et al. (2000). "The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?" EMBO reports 1(4): 319-322.
Jazayeri, A., J. Falck, et al. (2006). "ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks." Nat Cell Biol 8(1): 37-45.
Jiang, W., D. McDonald, et al. (1999). "Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication." The EMBO journal 18(20): 5703-5713.
Kelly, T. J. and G. W. Brown (2000). "Regulation of chromosome replication." Annual review of biochemistry 69: 829-880.
Kim, J. S., T. B. Krasieva, et al. (2005). "Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells." J Cell Biol 170(3): 341-347.
Kim, S. T. (2005). "Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649." Biochem Biophys Res Commun 331(1): 247-252.
Kim, S. T. (2005). "Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649." Biochemical and biophysical research communications 331(1): 247-252.
Krogh, B. O. and L. S. Symington (2004). "Recombination proteins in yeast." Annu Rev Genet 38: 233-271.
Labib, K. (2010). "How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?" Genes & development 24(12): 1208-1219.
Lavin, M. F. and S. Kozlov (2007). "ATM activation and DNA damage response." Cell Cycle 6(8): 931-942.
Lee, J. H. and T. T. Paull (2004). "Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex." Science 304(5667): 93-96.
Lee, J. H. and T. T. Paull (2005). "ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex." Science 308(5721): 551-554.
Lee, J. H. and T. T. Paull (2007). "Activation and regulation of ATM kinase activity in response to DNA double-strand breaks." Oncogene 26(56): 7741-7748.
Lei, M. and B. K. Tye (2001). "Initiating DNA synthesis: from recruiting to activating the MCM complex." Journal of cell science 114(Pt 8): 1447-1454.
Lei, M. and B. K. Tye (2001). "Initiating DNA synthesis: from recruiting to activating the MCM complex." J Cell Sci 114(Pt 8): 1447-1454.
Lempiainen, H. and T. D. Halazonetis (2009). "Emerging common themes in regulation of PIKKs and PI3Ks." EMBO J 28(20): 3067-3073.
Levitzki, A. and S. Klein (2010). "Signal transduction therapy of cancer." Molecular aspects of medicine 31(4): 287-329.
Lim, D. S., S. T. Kim, et al. (2000). "ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway." Nature 404(6778): 613-617.
Liu, E., A. Y. Lee, et al. (2007). "The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted." J Cell Biol 179(4): 643-657.
Lukas, J., C. Lukas, et al. (2011). "More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance." Nat Cell Biol 13(10): 1161-1169.
Mao, Z., M. Bozzella, et al. (2008). "Comparison of nonhomologous end joining and homologous recombination in human cells." DNA Repair (Amst) 7(10): 1765-1771.
Masai, H. and K. Arai (2002). "Cdc7 kinase complex: a key regulator in the initiation of DNA replication." J Cell Physiol 190(3): 287-296.
Masutomi, K., E. Y. Yu, et al. (2003). "Telomerase maintains telomere structure in normal human cells." Cell 114(2): 241-253.
Matsuoka, S., B. A. Ballif, et al. (2007). "ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage." Science 316(5828): 1160-1166.
McKerlie, M., J. R. Walker, et al. (2013). "Phosphorylated (pT371)TRF1 is recruited to sites of DNA damage to facilitate homologous recombination and checkpoint activation." Nucleic Acids Res.
Melander, F., S. Bekker-Jensen, et al. (2008). "Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage-modified chromatin." J Cell Biol 181(2): 213-226.
Montagnoli, A., J. Moll, et al. (2010). "Targeting cell division cycle 7 kinase: a new approach for cancer therapy." Clin Cancer Res 16(18): 4503-4508.
Mu, J. J., Y. Wang, et al. (2007). "A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints." The Journal of biological chemistry 282(24): 17330-17334.
Pasero, P., B. P. Duncker, et al. (1999). "A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication." Genes & development 13(16): 2159-2176.
Peasland, A., L. Z. Wang, et al. (2011). "Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines." Br J Cancer 105(3): 372-381.
Reinhardt, H. C. and M. B. Yaffe (2009). "Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2." Curr Opin Cell Biol 21(2): 245-255.
Riballo, E., M. Kuhne, et al. (2004). "A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci." Mol Cell 16(5): 715-724.
Robison, J. G., J. Elliott, et al. (2004). "Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks." J Biol Chem 279(33): 34802-34810.
Rodriguez, R. and M. Meuth (2006). "Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress." Mol Biol Cell 17(1): 402-412.
Rothstein, R., B. Michel, et al. (2000). "Replication fork pausing and recombination or "gimme a break"." Genes Dev 14(1): 1-10.
Schild, D. and C. Wiese (2010). "Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability." Nucleic Acids Res 38(4): 1061-1070.
Sclafani, R. A. and T. M. Holzen (2007). "Cell cycle regulation of DNA replication." Annual review of genetics 41: 237-280.
Shell, S. M., Z. Li, et al. (2009). "Checkpoint kinase ATR promotes nucleotide excision repair of UV-induced DNA damage via physical interaction with xeroderma pigmentosum group A." J Biol Chem 284(36): 24213-24222.
Shiloh, Y. (2003). "ATM and related protein kinases: safeguarding genome integrity." Nat Rev Cancer 3(3): 155-168.
Stokes, M. P., J. Rush, et al. (2007). "Profiling of UV-induced ATM/ATR signaling pathways." Proceedings of the National Academy of Sciences of the United States of America 104(50): 19855-19860.
Summers, K. C., F. Shen, et al. (2011). "Phosphorylation: the molecular switch of double-strand break repair." Int J Proteomics 2011: 373816.
Tsuji, T., E. Lau, et al. (2008). "The role of Dbf4/Drf1-dependent kinase Cdc7 in DNA-damage checkpoint control." Molecular cell 32(6): 862-869.
Uziel, T., Y. Lerenthal, et al. (2003). "Requirement of the MRN complex for ATM activation by DNA damage." EMBO J 22(20): 5612-5621.
Vassin, V. M., M. S. Wold, et al. (2004). "Replication protein A (RPA) phosphorylation prevents RPA association with replication centers." Mol Cell Biol 24(5): 1930-1943.
Williams, G. J., S. P. Lees-Miller, et al. (2010). "Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks." DNA Repair (Amst) 9(12): 1299-1306.
Williams, R. S. and J. A. Tainer (2007). "Learning our ABCs: Rad50 directs MRN repair functions via adenylate kinase activity from the conserved ATP binding cassette." Mol Cell 25(6): 789-791.
Williams, R. S., J. S. Williams, et al. (2007). "Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template." Biochemistry and cell biology = Biochimie et biologie cellulaire 85(4): 509-520.
Wold, M. S. (1997). "Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism." Annu Rev Biochem 66: 61-92.
Wu, X., V. Ranganathan, et al. (2000). "ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response." Nature 405(6785): 477-482.
Wyman, C. and R. Kanaar (2006). "DNA double-strand break repair: all's well that ends well." Annual review of genetics 40: 363-383.
Yoon, H. J., S. Loo, et al. (1993). "Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle." Molecular biology of the cell 4(2): 195-208.
Yuan, J., R. Adamski, et al. (2010). "Focus on histone variant H2AX: to be or not to be." FEBS Lett 584(17): 3717-3724.
Zakikhani, M., M. Bazile, et al. (2012). "Alterations in cellular energy metabolism associated with the antiproliferative effects of the ATM inhibitor KU-55933 and with metformin." PLoS One 7(11): e49513.
Zhao, S., Y. C. Weng, et al. (2000). "Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products." Nature 405(6785): 473-477.
Zou, L. and S. J. Elledge (2003). "Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes." Science 300(5625): 1542-1548.