去氧核醣核苷三磷酸 (dNTP)的生合成在細胞中受到嚴密的調控，並且對於去氧核醣核酸 (DNA)的複製與修復是重要的。當細胞中的dNTP的調控不受到控制時，往往伴隨著不正常的生理狀態，尤其是其中的胸腺嘧啶三磷酸(dTTP)。dTTP的生合成是透過新生成(de novo)與回收(salvage)的路徑，形成胸腺嘧啶單磷酸(dTMP)後，再藉由胸腺嘧啶單磷酸激酶(TMPK)磷酸化成胸腺嘧啶雙磷酸(dTDP)，最後經由雙核酸激酶(NDPK)形成dTTP。
由於dTTP的生成無論是透過新生成或是再回收的方式都必須透過TMPK調控，TMPK也因此被認為是個有潛力的標靶來治療細菌感染。然而，TMPK抑制劑還尚未應用在治療真菌感染方面。白色念珠菌是主要的院內感染的真菌，在比較了白色念珠菌的TMPK跟人類TMPK的蛋白結構、催化活性、受質專一性等方面的差異性顯示:白色念珠菌的TMPK有一個獨特的環狀結構，而這個結構與它的高酵素活性以及磷酸化去氧尿嘧啶單磷酸(dUMP)的能力有關。在酵母菌中表現白色念珠菌的TMPK相較於表現人類的TMPK會對的5 -氟尿嘧啶 (5-FU)與5 -氟尿嘧啶核苷(5-Fluorouridine, 5-FUrd)較敏感。進一步利用CRISPR編輯技術在白色念珠菌中刪除TMPK該環狀結構的基因序列，不但會嚴重減緩菌株的生長，也會降低對於嘧啶類似物的藥物敏感性。與此發現一致地是，5-FUrd對於致病性以及抗藥性的白色念珠菌皆有毒殺性，可以做為抗真菌藥物的新選項。最後，利用四環黴素的基因抑制系統證明了TMPK在白色念珠菌中是個生長所需的蛋白，能成為研發抗真菌藥物的新標靶。
另外，在本論文研究中，我發展一個新的方法來測量細胞內的dNTP含量。當細胞處於在不正常的生理條件下時，細胞內dNTP的含量並不會維持恆定;許多臨床化療藥物機制是透過影響核苷酸的生合成與基因損傷，因此，測量細胞內dNTP含量可以提供細胞的生長狀況與藥物反應的訊息。以往需要使用放射性同位素及高效液相層析(用紫外線偵測或是搭配質譜偵測)(HPLC-UV or HPLC/MS/MS)來測量細胞內dNTP的含量。這個新方法是利用銅催化疊氮與炔烃環加成反應（CuAAC）將螢光染劑標記在帶有炔烃的新生成DNA上，並用微量盤式分析儀測量螢光數值。使用這個新方法與放射性同位素的方法測細胞內的dNTP可以得到類似的結果，顯示我發展的實驗方法可以取代傳統放射性偵測的方式。而這個方法同時可用96微孔盤增加分析量，用於測量不同藥物處理後，細胞內dNTP量的變化。

Intracellular supplement of deoxyribonucleoside triphosphate (dNTP) is under highly regulated processes and is important for genome integrity. Dysregulation of cellular dNTP level is associated with abnormal physiological conditions, especially thymidine triphosphate (dTTP) pool. The synthesis of dTTP is mediated by both de novo and salvage pathways, by which thymidine monophosphate (dTMP) synthesized. dTMP is further phosphorylated to thymidine diphosphate (dTDP) and dTTP through thymidylate kinase (TMPK) and nucleoside diphosphate kinase (NDPK), respectively.
Being the key enzyme in catalyzing the merged pathway of dTTP synthesis, TMPK is well studied as a drug target in many bacterial infections. However, the activity of TMPK inhibitor on antifungal therapy has not been persuaded. Here, I investigated the differences in protein structures, kinetic properties, and substrate specificity between TMPKs that from Homo sapiens (hTMPK) and Candida albicans (CaTMPK), the major fungal pathogens in healthcare-associated infections, and identified a unique loop in CaTMPK that confers hyperactivity and enables the utilization of deoxyuridine monophosphate (dUMP) over hTMPK. Compared with hTMPK, expression of CaTMPK in S. cerevisiae produces more cytotoxic effect of 5-Fluorouracil (5-FU)/ 5-Fluorouridine (5-FUrd). CRISPR-mediated deletion of this loop in C. albicans significantly reduces the growth rate and the susceptibility to pyrimidine analogues. Furthermore, I found 5-fluorouridine (5-FUrd) as a potential anti-fungal that is toxic to both pathogenic and drug resistance C. albicans. In this end, I demonstrated the essentiality of CaTMPK in C. albicans by using repression system. Thus, CaTMPK is a proved target for antifungal reagents development.
In this thesis, I also developed a new method to quantify cellular dNTP pools. When cells under physiological conditions, the levels of dNTP pools are changed. Many chemotherapy drugs disrupt dNTP/rNTP synthesis and cause genome toxicity. Hence, quantification of cellular dNTP pools can provide information about the condition of cell growth or the response to drug treatments. Both of isotopes-based and HPLC-UV or HPLC/MS/MS methods are current methods for dNTP quantitation. The method I developed is based on copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction to label alkyne-modified DNA with fluorescent dyes for the detection of fluorescence unit by a ELISA reader. I proved that click method is comparable to current isotope-based method on cellular dNTP quantification, suggesting that the new method could replace the old one. Moreover, I applied the magnetic separation to modify this assay in a 96-well microplate format and successfully quantitated the alteration of cellular dNTP pools in response to different drug treatments.
 

中文摘要 i
Abstract ii
Table of contents iii
Chapter 1. Overview and rational 1
1-1. Pathogenicity and drug resistance in Candida albicans 2
1-1.1. Candida albicans is the common fungal pathogen in healthcare-associated infection 2
1-1.2. The mechanisms of antifungal drugs 2
1-1.3. Fungal drug resistance 3
1-2. Overview of Thymidylate kinase 5
1-2.1. The role of thymidylate kinase in dTTP synthesis 5
1-2.2. Structures of TMPKs 5
1-2.3. Substrate selectivity of TMPKs 6
1-3. Deoxyribonucleoside triphosphates metabolism, imbalance and cancer 9
1-3.1. Biosynthesis of deoxyribonucleoside triphosphates (dNTPs) 9
1-3.2. dNTP level and genome instability 10
1-4. Current methods for cellular dNTP quantification 11
1-4.1. Enzymatic method 11
1-4.2. High-performance liquid chromatography (HPLC)-based assay and HPLC coupled liquid chromatography–tandem mass spectrometry (LC/MS/MS) 11
Chapter 2. The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans 13
Abstract 14
Introduction 15
Materials and methods 17
Results 22
Discussion 27
List of figures 29
Chapter 3. Quantitation of deoxynucleoside triphosphates by combining enzymatic and click reactions 53
Abstract 54
Introduction 55
Results 60
Discussion 64
List of figures 66
Perspective 83
Reference 86
Appendix 96

Aird, K. M., G. Zhang, H. Li, Z. Tu, B. G. Bitler, A. Garipov, H. Wu, Z. Wei, S. N. Wagner, M. Herlyn, and R. Zhang. 2013. 'Suppression of nucleotide metabolism underlies the establishment and maintenance of oncogene-induced senescence', Cell Rep, 3: 1252-65.
Albertson, G. D., M. Niimi, R. D. Cannon, and H. F. Jenkinson. 1996. 'Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance', Antimicrob Agents Chemother, 40: 2835-41.
Aye, Y., M. Li, M. J. Long, and R. S. Weiss. 2015. 'Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies', Oncogene, 34: 2011-21.
Belenky, P., D. Camacho, and J. J. Collins. 2013. 'Fungicidal drugs induce a common oxidative-damage cellular death pathway', Cell Rep, 3: 350-8.
Berlin, J., and A. B. Benson, 3rd. 2010. 'Chemotherapy: Gemcitabine remains the standard of care for pancreatic cancer', Nat Rev Clin Oncol, 7: 135-7.
Bester, A. C., M. Roniger, Y. S. Oren, M. M. Im, D. Sarni, M. Chaoat, A. Bensimon, G. Zamir, D. S. Shewach, and B. Kerem. 2011. 'Nucleotide deficiency promotes genomic instability in early stages of cancer development', Cell, 145: 435-46.
Birkenmeyer, L. G., J. C. Hill, and L. B. Dumas. 1984. 'Saccharomyces cerevisiae CDC8 gene and its product', Mol Cell Biol, 4: 583-90.
Bogenhagen, D., and D. A. Clayton. 1976. 'Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells', J Biol Chem, 251: 2938-44.
Branco, J., M. Ola, R. M. Silva, E. Fonseca, N. C. Gomes, C. Martins-Cruz, A. P. Silva, A. Silva-Dias, C. Pina-Vaz, C. Erraught, L. Brennan, A. G. Rodrigues, G. Butler, and I. M. Miranda. 2017. 'Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance', Clin Microbiol Infect, 23: 575 e1-75 e8.
Brown, K. K., J. B. Spinelli, J. M. Asara, and A. Toker. 2017. 'Adaptive Reprogramming of De Novo Pyrimidine Synthesis Is a Metabolic Vulnerability in Triple-Negative Breast Cancer', Cancer Discov, 7: 391-99.
Buj, R., and K. M. Aird. 2018. 'Deoxyribonucleotide Triphosphate Metabolism in Cancer and Metabolic Disease', Front Endocrinol (Lausanne), 9: 177.
Cahova, H., A. Panattoni, P. Kielkowski, J. Fanfrlik, and M. Hocek. 2016. '5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs', ACS Chem Biol, 11: 3165-71.
Chabosseau, P., G. Buhagiar-Labarchede, R. Onclercq-Delic, S. Lambert, M. Debatisse, O. Brison, and M. Amor-Gueret. 2011. 'Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome', Nat Commun, 2: 368.
Chang, Z. F., D. Y. Huang, and N. C. Hsue. 1994. 'Differential phosphorylation of human thymidine kinase in proliferating and M phase-arrested human cells', J Biol Chem, 269: 21249-54.
Chen, C. W., N. Tsao, L. Y. Huang, Y. Yen, X. Liu, C. Lehman, Y. H. Wang, M. C. Tseng, Y. J. Chen, Y. C. Ho, C. F. Chen, and Z. F. Chang. 2016. 'The Impact of dUTPase on Ribonucleotide Reductase-Induced Genome Instability in Cancer Cells', Cell Rep, 16: 1287-99.
Chen, Y. C., S. F. Kuo, F. J. Chen, and C. H. Lee. 2017. 'Antifungal susceptibility of Candida species isolated from patients with candidemia in southern Taiwan, 2007-2012: impact of new antifungal breakpoints', Mycoses, 60: 89-95.
Chen, Y. H., H. Y. Hsu, M. T. Yeh, C. C. Chen, C. Y. Huang, Y. H. Chung, Z. F. Chang, W. C. Kuo, N. L. Chan, J. H. Weng, B. C. Chung, Y. J. Chen, C. B. Jian, C. C. Shen, H. C. Tai, S. Y. Sheu, and J. M. Fang. 2016. 'Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation', J Med Chem, 59: 9906-18.
"Clinical and Laboratory Standards Institute. Epidemiological cutoff values for antifungal susceptibility testing. 1st ed., CLSI standard M59." In. 2016. Clinical and Laboratory Standards Institute, Wayne, PA, USA.
"Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts, 4th ed. CLSI standard M27." In. 2017. Clinical and Laboratory Standards Institute, Wayne, PA, USA.
Cowen, L. E. 2008. 'The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype', Nat Rev Microbiol, 6: 187-98.
Cowen, L. E., and W. J. Steinbach. 2008. 'Stress, drugs, and evolution: the role of cellular signaling in fungal drug resistance', Eukaryot Cell, 7: 747-64.
Cui, H., L. M. Ruiz-Perez, D. Gonzalez-Pacanowska, and I. H. Gilbert. 2010. 'Potential application of thymidylate kinase in nucleoside analogue activation in Plasmodium falciparum', Bioorg Med Chem, 18: 7302-9.
Cui, Q., W. S. Shin, Y. Luo, J. Tian, H. Cui, and D. Yin. 2013. 'Thymidylate kinase: an old topic brings new perspectives', Curr Med Chem, 20: 1286-305.
Davidson, M. B., Y. Katou, A. Keszthelyi, T. L. Sing, T. Xia, J. Ou, J. A. Vaisica, N. Thevakumaran, L. Marjavaara, C. L. Myers, A. Chabes, K. Shirahige, and G. W. Brown. 2012. 'Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype', EMBO J, 31: 895-907.
Decosterd, L. A., E. Cottin, X. Chen, F. Lejeune, R. O. Mirimanoff, J. Biollaz, and P. A. Coucke. 1999. 'Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquid chromatography', Anal Biochem, 270: 59-68.
Di Pierro, D., B. Tavazzi, C. F. Perno, M. Bartolini, E. Balestra, R. Calio, B. Giardina, and G. Lazzarino. 1995. 'An ion-pairing high-performance liquid chromatographic method for the direct simultaneous determination of nucleotides, deoxynucleotides, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts', Anal Biochem, 231: 407-12.
Erbs, P., E. Regulier, J. Kintz, P. Leroy, Y. Poitevin, F. Exinger, R. Jund, and M. Mehtali. 2000. 'In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene', Cancer Res, 60: 3813-22.
Ferraro, P., E. Franzolin, G. Pontarin, P. Reichard, and V. Bianchi. 2010. 'Quantitation of cellular deoxynucleoside triphosphates', Nucleic Acids Res, 38: e85.
Fleck, O., R. Vejrup-Hansen, A. Watson, A. M. Carr, O. Nielsen, and C. Holmberg. 2013. 'Spd1 accumulation causes genome instability independently of ribonucleotide reductase activity but functions to protect the genome when deoxynucleotide pools are elevated', J Cell Sci, 126: 4985-94.
Foekens, J. A., S. Romain, M. P. Look, P. M. Martin, and J. G. Klijn. 2001. 'Thymidine kinase and thymidylate synthase in advanced breast cancer: response to tamoxifen and chemotherapy', Cancer Res, 61: 1421-5.
Ford, C. B., J. M. Funt, D. Abbey, L. Issi, C. Guiducci, D. A. Martinez, T. Delorey, B. Y. Li, T. C. White, C. Cuomo, R. P. Rao, J. Berman, D. A. Thompson, and A. Regev. 2015. 'The evolution of drug resistance in clinical isolates of Candida albicans', Elife, 4: e00662.
Franzolin, E., G. Pontarin, C. Rampazzo, C. Miazzi, P. Ferraro, E. Palumbo, P. Reichard, and V. Bianchi. 2013. 'The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells', Proc Natl Acad Sci U S A, 110: 14272-7.
Gabriel, F., A. Sabra, S. El-Kirat-Chatel, S. Pujol, V. Fitton-Ouhabi, D. Brethes, K. Dementhon, I. Accoceberry, and T. Noel. 2014. 'Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole', Antimicrob Agents Chemother, 58: 4476-85.
Gardner, A. F., and W. E. Jack. 1999. 'Determinants of nucleotide sugar recognition in an archaeon DNA polymerase', Nucleic Acids Res, 27: 2545-53.
Gemble, S., A. Ahuja, G. Buhagiar-Labarchede, R. Onclercq-Delic, J. Dairou, D. S. Biard, S. Lambert, M. Lopes, and M. Amor-Gueret. 2015. 'Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA', PLoS Genet, 11: e1005384.
Gemble, S., G. Buhagiar-Labarchede, R. Onclercq-Delic, D. Biard, S. Lambert, and M. Amor-Gueret. 2016. 'A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation', J Cell Sci, 129: 3167-77.
Grem, J. L., K. D. Danenberg, K. Behan, A. Parr, L. Young, P. V. Danenberg, D. Nguyen, J. Drake, A. Monks, and C. J. Allegra. 2001. 'Thymidine kinase, thymidylate synthase, and dihydropyrimidine dehydrogenase profiles of cell lines of the National Cancer Institute's Anticancer Drug Screen', Clin Cancer Res, 7: 999-1009.
Guimaraes, A. P., F. R. de Souza, A. A. Oliveira, A. S. Goncalves, R. B. de Alencastro, T. C. Ramalho, and T. C. Franca. 2015. 'Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox', Eur J Med Chem, 91: 72-90.
Halazonetis, T. D., V. G. Gorgoulis, and J. Bartek. 2008. 'An oncogene-induced DNA damage model for cancer development', Science, 319: 1352-5.
Hargrove, T. Y., L. Friggeri, Z. Wawrzak, A. Qi, W. J. Hoekstra, R. J. Schotzinger, J. D. York, F. P. Guengerich, and G. I. Lepesheva. 2017. 'Structural analyses of Candida albicans sterol 14alpha-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis', J Biol Chem, 292: 6728-43.
Hartwell, L. H. 1971. 'Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation', J Mol Biol, 59: 183-94.
Hartwell, L. H., R. K. Mortimer, J. Culotti, and M. Culotti. 1973. 'Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants', Genetics, 74: 267-86.
Hastak, K., R. K. Paul, M. K. Agarwal, V. S. Thakur, A. R. Amin, S. Agrawal, R. M. Sramkoski, J. W. Jacobberger, M. W. Jackson, G. R. Stark, and M. L. Agarwal. 2008. 'DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation', Proc Natl Acad Sci U S A, 105: 6314-9.
Hennere, G., F. Becher, A. Pruvost, C. Goujard, J. Grassi, and H. Benech. 2003. 'Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals', J Chromatogr B Analyt Technol Biomed Life Sci, 789: 273-81.
Hills, S. A., and J. F. Diffley. 2014. 'DNA replication and oncogene-induced replicative stress', Curr Biol, 24: R435-44.
Hope, W. W., L. Tabernero, D. W. Denning, and M. J. Anderson. 2004. 'Molecular mechanisms of primary resistance to flucytosine in Candida albicans', Antimicrob Agents Chemother, 48: 4377-86.
Hu, C. M., M. T. Yeh, N. Tsao, C. W. Chen, Q. Z. Gao, C. Y. Chang, M. H. Lee, J. M. Fang, S. Y. Sheu, C. J. Lin, M. C. Tseng, Y. J. Chen, and Z. F. Chang. 2012. 'Tumor cells require thymidylate kinase to prevent dUTP incorporation during DNA repair', Cancer Cell, 22: 36-50.
Hu, R., L. Li, B. Degreve, G. E. Dutschman, W. Lam, and Y. C. Cheng. 2005. 'Behavior of Thymidylate Kinase toward Monophosphate Metabolites and Its Role in the Metabolism of 1-(2'-Deoxy-2'-Fluoro- -L-Arabinofuranosyl)-5-Methyluracil (Clevudine) and 2',3'-Didehydro-2',3'-Dideoxythymidine in Cells', Antimicrobial Agents and Chemotherapy, 49: 2044-49.
Huang, D., Y. Zhang, and X. Chen. 2003. 'Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography', J Chromatogr B Analyt Technol Biomed Life Sci, 784: 101-9.
Jager, S., G. Rasched, H. Kornreich-Leshem, M. Engeser, O. Thum, and M. Famulok. 2005. 'A versatile toolbox for variable DNA functionalization at high density', J Am Chem Soc, 127: 15071-82.
Kandeel, M., and Y. Kitade. 2008. 'Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase', J Biochem, 144: 245-50.
Kandeel, M., Y. Kitade, A. Al-Taher, and M. Al-Nazawi. 2019. 'The structural basis of unique substrate recognition by Plasmodium thymidylate kinase: Molecular dynamics simulation and inhibitory studies', PLoS One, 14: e0212065.
Ke, P. Y., Y. Y. Kuo, C. M. Hu, and Z. F. Chang. 2005. 'Control of dTTP pool size by anaphase promoting complex/cyclosome is essential for the maintenance of genetic stability', Genes Dev, 19: 1920-33.
Kohnken, R., K. M. Kodigepalli, and L. Wu. 2015. 'Regulation of deoxynucleotide metabolism in cancer: novel mechanisms and therapeutic implications', Mol Cancer, 14: 176.
Kong, Z., S. Jia, A. L. Chabes, P. Appelblad, R. Lundmark, T. Moritz, and A. Chabes. 2018. 'Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry', Nucleic Acids Res, 46: e66.
Kumar, D., J. Viberg, A. K. Nilsson, and A. Chabes. 2010. 'Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint', Nucleic Acids Res, 38: 3975-83.
Kuwahara, M., J. Nagashima, M. Hasegawa, T. Tamura, R. Kitagata, K. Hanawa, S. Hososhima, T. Kasamatsu, H. Ozaki, and H. Sawai. 2006. 'Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA', Nucleic Acids Res, 34: 5383-94.
Kyprianou, N., and J. T. Isaacs. 1989. '"Thymineless" death in androgen-independent prostatic cancer cells', Biochem Biophys Res Commun, 165: 73-81.
LaFayette, S. L., C. Collins, A. K. Zaas, W. A. Schell, M. Betancourt-Quiroz, A. A. Gunatilaka, J. R. Perfect, and L. E. Cowen. 2010. 'PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90', PLoS Pathog, 6: e1001069.
Lai, W. C., H. F. Sun, P. H. Lin, H. L. Ho Lin, and J. C. Shieh. 2016. 'A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans', Curr Genet, 62: 213-35.
Lavie, A., M. Konrad, R. Brundiers, R. S. Goody, I. Schlichting, and J. Reinstein. 1998. 'Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution: implications for catalysis and AZT activation', Biochemistry, 37: 3677-86.
Lavie, A., I. R. Vetter, M. Konrad, R. S. Goody, J. Reinstein, and I. Schlichting. 1997. 'Structure of thymidylate kinase reveals the cause behind the limiting step in AZT activation', Nat Struct Biol, 4: 601-4.
Liu, C., K. A. Powell, K. Mundt, L. Wu, A. M. Carr, and T. Caspari. 2003. 'Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms', Genes Dev, 17: 1130-40.
Lo, H. J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, and G. R. Fink. 1997. 'Nonfilamentous C. albicans mutants are avirulent', Cell, 90: 939-49.
Longley, D. B., D. P. Harkin, and P. G. Johnston. 2003. '5-fluorouracil: mechanisms of action and clinical strategies', Nat Rev Cancer, 3: 330-8.
Luna-Tapia, A., H. M. E. Willems, J. E. Parker, H. Tournu, K. S. Barker, A. T. Nishimoto, P. D. Rogers, S. L. Kelly, B. M. Peters, and G. E. Palmer. 2018. 'Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity', MBio, 9.
Lupetti, A., R. Danesi, M. Campa, M. Del Tacca, and S. Kelly. 2002. 'Molecular basis of resistance to azole antifungals', Trends Mol Med, 8: 76-81.
Mannava, S., K. C. Moparthy, L. J. Wheeler, V. Natarajan, S. N. Zucker, E. E. Fink, M. Im, S. Flanagan, W. C. Burhans, N. C. Zeitouni, D. S. Shewach, C. K. Mathews, and M. A. Nikiforov. 2013. 'Depletion of deoxyribonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene-induced senescence', Am J Pathol, 182: 142-51.
Martel, C. M., J. E. Parker, O. Bader, M. Weig, U. Gross, A. G. Warrilow, D. E. Kelly, and S. L. Kelly. 2010. 'A clinical isolate of Candida albicans with mutations in ERG11 (encoding sterol 14alpha-demethylase) and ERG5 (encoding C22 desaturase) is cross resistant to azoles and amphotericin B', Antimicrob Agents Chemother, 54: 3578-83.
Martel, C. M., J. E. Parker, O. Bader, M. Weig, U. Gross, A. G. Warrilow, N. Rolley, D. E. Kelly, and S. L. Kelly. 2010. 'Identification and characterization of four azole-resistant erg3 mutants of Candida albicans', Antimicrob Agents Chemother, 54: 4527-33.
Mathews, C. K. 2015. 'Deoxyribonucleotide metabolism, mutagenesis and cancer', Nat Rev Cancer, 15: 528-39.
Meuth, M. 1989. 'The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells', Exp Cell Res, 181: 305-16.
Mole, M. L., D. L. Hunter, P. Gao, and C. Lau. 1998. 'Sample preparation and high-performance liquid chromatographic analysis of deoxyribonucleoside triphosphates in individual rat embryos', Anal Biochem, 259: 245-52.
Munro, C. A. 2010. 'Fungal echinocandin resistance', F1000 Biol Rep, 2: 66.
Nakayama, H., T. Mio, S. Nagahashi, M. Kokado, M. Arisawa, and Y. Aoki. 2000. 'Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans', Infect Immun, 68: 6712-9.
Naumova, L. A., O. A. Aizenberg, and G. E. Fradkin. 1982. '[Effect of polB mutation on the nature of the thymineless death of thy- cells of Escherichia coli K-12]', Genetika, 18: 1408-11.
Nett, J. E., K. Crawford, K. Marchillo, and D. R. Andes. 2010. 'Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene', Antimicrob Agents Chemother, 54: 3505-8.
Nett, J. E., H. Sanchez, M. T. Cain, K. M. Ross, and D. R. Andes. 2011. 'Interface of Candida albicans biofilm matrix-associated drug resistance and cell wall integrity regulation', Eukaryot Cell, 10: 1660-9.
Nitiss, J. L. 2009. 'Targeting DNA topoisomerase II in cancer chemotherapy', Nat Rev Cancer, 9: 338-50.
Ostermann, N., I. Schlichting, R. Brundiers, M. Konrad, J. Reinstein, T. Veit, R. S. Goody, and A. Lavie. 2000. 'Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate', Structure, 8: 629-42.
Pappas, P. G., M. S. Lionakis, M. C. Arendrup, L. Ostrosky-Zeichner, and B. J. Kullberg. 2018. 'Invasive candidiasis', Nat Rev Dis Primers, 4: 18026.
Pierce, C. G., T. Vila, J. A. Romo, D. Montelongo-Jauregui, G. Wall, A. Ramasubramanian, and J. L. Lopez-Ribot. 2017. 'The Candida albicans Biofilm Matrix: Composition, Structure and Function', J Fungi (Basel), 3.
Polak, A., and W. H. Wain. 1979. 'The effect of 5-fluorocytosine on the blastospores and hyphae of Candida albicans', J Med Microbiol, 12: 83-97.
Pourakbari, B., M. Teymuri, S. Mahmoudi, S. K. Valian, Z. Movahedi, H. Eshaghi, and S. Mamishi. 2017. 'Expression of Major Efflux Pumps in Fluconazole-Resistant Candida albicans', Infect Disord Drug Targets.
Prakash, S., and L. Prakash. 2002. 'Translesion DNA synthesis in eukaryotes: a one- or two-polymerase affair', Genes Dev, 16: 1872-83.
Revie, N. M., K. R. Iyer, N. Robbins, and L. E. Cowen. 2018. 'Antifungal drug resistance: evolution, mechanisms and impact', Curr Opin Microbiol, 45: 70-76.
Rocha, M. F., S. P. Bandeira, L. P. de Alencar, L. M. Melo, J. A. Sales, M. A. Paiva, C. E. Teixeira, D. S. Castelo-Branco, W. A. Pereira-Neto, R. A. Cordeiro, J. J. Sidrim, and R. S. Brilhante. 2017. 'Azole resistance in Candida albicans from animals: Highlights on efflux pump activity and gene overexpression', Mycoses.
Sanglard, D., A. Coste, and S. Ferrari. 2009. 'Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation', FEMS Yeast Res, 9: 1029-50.
Santi, D. V., and C. S. McHenry. 1972. '5-Fluoro-2'-deoxyuridylate: covalent complex with thymidylate synthetase', Proc Natl Acad Sci U S A, 69: 1855-7.
Sato, T., A. Neschadim, M. Konrad, D. H. Fowler, A. Lavie, and J. A. Medin. 2007. 'Engineered human tmpk/AZT as a novel enzyme/prodrug axis for suicide gene therapy', Mol Ther, 15: 962-70.
Seiple, L., P. Jaruga, M. Dizdaroglu, and J. T. Stivers. 2006. 'Linking uracil base excision repair and 5-fluorouracil toxicity in yeast', Nucleic Acids Res, 34: 140-51.
Selmecki, A. M., K. Dulmage, L. E. Cowen, J. B. Anderson, and J. Berman. 2009. 'Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance', PLoS Genet, 5: e1000705.
Sherman, P. A., and J. A. Fyfe. 1989. 'Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers', Anal Biochem, 180: 222-6.
Shor, E., and D. S. Perlin. 2015. 'Coping with stress and the emergence of multidrug resistance in fungi', PLoS Pathog, 11: e1004668.
Singh, A., and Y. J. Xu. 2016. 'The Cell Killing Mechanisms of Hydroxyurea', Genes (Basel), 7.
Sinha, K., and G. S. Rule. 2017. 'The Structure of Thymidylate Kinase from Candida albicans Reveals a Unique Structural Element', Biochemistry, 56: 4360-70.
Su, H., L. Han, and X. Huang. 2018. 'Potential targets for the development of new antifungal drugs', J Antibiot (Tokyo).
Su, J. Y., and R. A. Sclafani. 1991. 'Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast', Nucleic Acids Res, 19: 823-7.
Sudbery, P. E. 2011. 'Growth of Candida albicans hyphae', Nat Rev Microbiol, 9: 737-48.
Sullivan, D. J., G. P. Moran, E. Pinjon, A. Al-Mosaid, C. Stokes, C. Vaughan, and D. C. Coleman. 2004. 'Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans', FEMS Yeast Res, 4: 369-76.
Taff, H. T., K. F. Mitchell, J. A. Edward, and D. R. Andes. 2013. 'Mechanisms of Candida biofilm drug resistance', Future Microbiol, 8: 1325-37.
Tanaka, K., A. Yoshioka, S. Tanaka, and Y. Wataya. 1984. 'An improved method for the quantitative determination of deoxyribonucleoside triphosphates in cell extracts', Anal Biochem, 139: 35-41.
Techer, H., S. Koundrioukoff, A. Nicolas, and M. Debatisse. 2017. 'The impact of replication stress on replication dynamics and DNA damage in vertebrate cells', Nat Rev Genet, 18: 535-50.
Thakur, J. K., H. Arthanari, F. Yang, S. J. Pan, X. Fan, J. Breger, D. P. Frueh, K. Gulshan, D. K. Li, E. Mylonakis, K. Struhl, W. S. Moye-Rowley, B. P. Cormack, G. Wagner, and A. M. Naar. 2008. 'A nuclear receptor-like pathway regulating multidrug resistance in fungi', Nature, 452: 604-9.
Toussaint, M., I. Dionne, and R. J. Wellinger. 2005. 'Limited TTP supply affects telomere length regulation in a telomerase-independent fashion', Nucleic Acids Res, 33: 704-13.
Turner, S. A., and G. Butler. 2014. 'The Candida pathogenic species complex', Cold Spring Harb Perspect Med, 4: a019778.
Ullman, B., S. M. Clift, L. J. Gudas, B. B. Levinson, M. A. Wormsted, and D. W. Martin, Jr. 1980. 'Alterations in deoxyribonucleotide metabolism in cultured cells with ribonucleotide reductase activities refractory to feedback inhibition by 2'-deoxyadenosine triphosphate', J Biol Chem, 255: 8308-14.
Vale-Silva, L. A., A. T. Coste, F. Ischer, J. E. Parker, S. L. Kelly, E. Pinto, and D. Sanglard. 2012. 'Azole resistance by loss of function of the sterol Delta(5),(6)-desaturase gene (ERG3) in Candida albicans does not necessarily decrease virulence', Antimicrob Agents Chemother, 56: 1960-8.
Vickers, M. F., S. Y. Yao, S. A. Baldwin, J. D. Young, and C. E. Cass. 2000. 'Nucleoside transporter proteins of Saccharomyces cerevisiae. Demonstration of a transporter (FUI1) with high uridine selectivity in plasma membranes and a transporter (FUN26) with broad nucleoside selectivity in intracellular membranes', J Biol Chem, 275: 25931-8.
Vyas, V. K., M. I. Barrasa, and G. R. Fink. 2015. 'A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families', Sci Adv, 1: e1500248.
Walker, L. A., N. A. Gow, and C. A. Munro. 2010. 'Fungal echinocandin resistance', Fungal Genet Biol, 47: 117-26.
Walther, A., and J. Wendland. 2003. 'An improved transformation protocol for the human fungal pathogen Candida albicans', Curr Genet, 42: 339-43.
Williams, J. S., S. A. Lujan, and T. A. Kunkel. 2016. 'Processing ribonucleotides incorporated during eukaryotic DNA replication', Nat Rev Mol Cell Biol, 17: 350-63.
Wilson, P. M., M. J. Labonte, J. Russell, S. Louie, A. A. Ghobrial, and R. D. Ladner. 2011. 'A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates', Nucleic Acids Res, 39: e112.
Xu, D., B. Jiang, T. Ketela, S. Lemieux, K. Veillette, N. Martel, J. Davison, S. Sillaots, S. Trosok, C. Bachewich, H. Bussey, P. Youngman, and T. Roemer. 2007. 'Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans', PLoS Pathog, 3: e92.
You, L., W. Qian, Q. Yang, L. Mao, L. Zhu, X. Huang, J. Jin, and H. Meng. 2017. 'ERG11 gene mutations and MDR1 up-regulation confer pan-azole-resistance in Candida tropicalis causing disseminated candidiasis while on posaconazole prophylaxis in an acute lymphoblastic leukemia patient', Antimicrob Agents Chemother.
Zhang, B. N., A. Bueno Venegas, I. D. Hickson, and W. K. Chu. 2018. 'DNA replication stress and its impact on chromosome segregation and tumorigenesis', Semin Cancer Biol.
Zhang, W., S. Tan, E. Paintsil, G. E. Dutschman, E. A. Gullen, E. Chu, and Y. C. Cheng. 2011. 'Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique', Biochem Pharmacol, 82: 411-7.