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Probing the binding of interstrand crosslinked DNA containing a fluorescent base analog with human and E. coli homologs of O6-alkyl-guanine-DNA-alkyltransferase


Probing the binding of interstrand crosslinked DNA containing a fluorescent base analog with human and E. coli homologs of O6-alkyl-guanine-DNA-alkyltransferase

Vergara, Jordan (2012) Probing the binding of interstrand crosslinked DNA containing a fluorescent base analog with human and E. coli homologs of O6-alkyl-guanine-DNA-alkyltransferase. Masters thesis, Concordia University.

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O6-alkylguanine-DNA-alkyltransferase (AGT) has been implicated in reducing the therapeutic efficacy of alkylating agents that cause mutations and apoptosis. Certain chemotherapeutic treatments involve the use of alkylating agents; bis-chloroethylnitrosourea (BCNU) is an example of an agent that has been investigated which has been shown to introduce DNA interstrand crosslinks (ICL). AGT directly repairs O6-alkylated guanines by flipping the damaged base into the active site and irreversibly transferring the alkyl group to an active site cysteine. Previous studies revealed that human AGT could repair DNA containing an O6-2’-deoxyguanosine-heptylene-O6-2’-deoxyguanosine (O6-dG-heptylene-O6-dG) ICL in a 5’-GNC-3’ motif mimicking a hepsulfam lesion. It is not yet understood, however, how the enzyme could access the bulky ICL lesion.
We synthesized and studied DNA duplexes containing O6-dG-heptylene-O6-dG ICL lesion in a 5’-GNC-3’ motif (where N is any base) and a fluorescent base incorporated at various positions to observe the extent to which DNA is denatured upon AGT binding. 6-Methyl-3-(β-D-2-deoxyribofuranosyl)pyrrolo[2,3-d] pyrimidin-2-one (pyrrolo-dC), is a base analog that fluoresces when not base-paired and exhibits little effect on DNA stability, structure and AGT repair. All DNA substrates were synthesized and AGT homologs purified and characterized using various biophysical techniques including CD, UV spectroscopy, gel electrophoresis and MS-Q-TOF. Radioactivity binding and repair assays were used to characterize ICL-AGT association. The DNA substrates exhibited B-form structure regardless of pyrrolo-dC or ICL incorporation. Furthermore, C145S and R128A variants of AGT and wild type Ada-C (from E. coli) display no repair of the ICL DNA. The arginine “finger” (R128) appears to play an essential role in repair but not in damage detection. Incorporation of pyrrolo-dC did not specifically demonstrate the extent of DNA denaturation due to the observation of increased fluorescence when positioned near the end of the DNA double helix relative to when placed in the center of the duplex. Fluorescence studies provide a base with which to develop new AGT variants with more complex ICL DNA substrates to study their interaction and possibly provide a new method for determining binding dissociation constants without hazardous radioactive material. Better understanding of the interaction of AGT and its homologs with ICL DNA provides greater knowledge about AGT function, an enzyme that plays a role in both restoring genomic integrity and therapeutic resistance.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Vergara, Jordan
Institution:Concordia University
Degree Name:M. Sc.
Date:October 2012
Thesis Supervisor(s):Wilds, Christopher
ID Code:975056
Deposited On:13 Jun 2013 19:48
Last Modified:18 Jan 2018 17:39


1. Lindahl T. (1993) “Instability and decay of the primary structure of DNA.” Nature 362:709-715.
2. Noll DM, Mason TM, Miller PS. (2006). “Formation and repair of interstrand cross-links in DNA.” Chem Rev. 106:277-301.
3. Friedberg EC. (1995). “Out of the shadows and into the light: the emergence of DNA repair.” Trends Biochem Sci. 20:381.
4. Lawley PD, Brookes P. (1968). “Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids.” Biochem J. 109(3):433-447.
5. Rupert CS. (1962). “Photoenzymatic repair of ultraviolet damage in DNA. II. Formation of enzyme-substrate complex.” J Gen Physiol. 45:725-741.
6. Bender K, Federwisch M, Loggen U, Nehls P, Rajewsky MF. (1996). “Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context.” Nucleic Acids Res. 24:2087-2094.
7. Brent TP, Houghton PJ, Houghton JA. (1985). "O6-Alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea." Proc Natl Acad Sci USA. 82: 2985-2989.
8. Daniels DS, Mol CD, Arvai AS, Kanugula S, Pegg AE, Tainer JA. (2000). "Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding." EMBO 19: 1719–1730
9. Daniels DS, Woo TT, Luu KX, Noll DM, Clarke ND, Pegg AE, Tainer JA. (2004). "DNA binding and nucleotide flipping by the human DNA repair protein AGT." Nat Struct Mol Biol. 11: 714-720.
10. Dolan ME, Moschel RC, Pegg AE. (1990). “Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents.” Proc. Natl. Acad. Sci. 87: 5368-5372.
11. Drabløs F, Feyzi E, Aas PA, Vaagbø CB, Kavli B, Bratlie MS, Peña-Diaz J, Otterlei M, Slupphaug G, Krokan HE. (2004). “Alkylation damage in DNA and RNA-repair mechanisms and medical significance.” DNA Repair 3:1389-1407.
12. Duguid EM, Mishina Y., He C. (2003). "How do DNA repair proteins locate potential base lesions? A chemical crosslinking method to investigate O6-alkylguanine-DNA alkyltransferases." Chem Biol. 10: 827-835.
13. Duguid EM, Rice PA, He C. (2005). "The structure of the human AGT protein bound to DNA and its implications for damage detection." J Mol Biol. 350: 657-666.
14. Fang Q, Kanugula S., Pegg AE. (2005). "Function of domains of human O6-alkylguanine-DNA alkyltransferase." Biochemistry. 44: 15396-15405.
15. Fang Q, Noronha A., Murphy SP, Wilds CJ, Tubbs JL, Tainer JA, Chowdhury G, Guengerich FP, Pegg AE. (2008). "Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase." Biochemistry 47: 10892-10903.
16. Gerson SL, Willson JK. (1995). “O6-alkylguanine-DNA alkyltransferase. A target for the modulation of drug resistance.” Hematol Oncol Clin North Am. 9:431-450.
17. Gerson SL. (2002). "Clinical relevance of MGMT in treatment of cancer." J. Clin. Oncol. 20:2388-2399.
18. Goodtzova K, Kanugula S, Edara S, Pauly GT, Moschel RC, Pegg AE. (1997). “Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases.” J Biol Chem. 272:8332-8339.
19. Graves RJ, Li BF, Swann PF. (1989). “Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase.” Carcinogenesis. 10:661-666.
20. Guengerich FP, Fang Q, Liu L, Hachley DL, Pegg AE. (2003). “O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145.” Biochemistry 42:10965-10970.
21. Horiguchi M, Kim J, Matsunaga N, Kaji H, Egawa T, Makino K, Koyanagi S, Ohdo S. (2010). “Glucocorticoid-dependent expression of O6-methylguanine-DNA methyltransferase gene modulates dacarbazine-induced hepatotoxicity in mice.” J. Pharmacol. Exp. Ther. 333:782-787.
22. Hu J, Ma A, Dinner AR. (2008). “A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT.” Proc. Natl. Acad. Sci. U.S.A. 105:4615-4620.
23. Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K. (2003). “Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo.” Chem Biol. 10:313-317.
24. Kaina B, Fritz G, Mitra S, Coquerelle T. (1991). "Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.." Carcinogenesis. 12: 1857-1867.
25. Kanugula S, Goodtzova K, Pegg AE. (1998). "Probing of conformational changes in human O6-alkylguanine-DNA alkyl transferase protein in its alkylated and DNA-bound states by limited proteolysis." Biochem J. 329: 545-550.
26. Kanugula S, Pegg AE. (2001). “Novel DNA repair alkyltransferase from Caenorhabditis elegans.” Environ Mol Mutagen. 38:235-243.
27. Kanugula S, Pauly GT, Moschel RC, Pegg AE. (2005). “A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities.” Proc Natl Acad Sci USA. 102:3617-3622.
28. Kleibl K. (2002). “Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O6-methylguanine DNA-methyltransferase in other organisms.” Mutat Res. 512:67-84.
29. Klein S, Oesch F. (1992). “Assay for O6-alkylguanine-DNA-alkyltransferase using oligonucleotides containing O6-methylguanine in a BamHI recognition site as substrate.” Anal Biochem. 205:294-299.
30. Kotandeniya D, Murphy D, Seneviratne U, Guza R, Pegg A, Kanugula S, Tretyakova N. (2011). “Mass spectrometry based approach to study the kinetics of O6-alkylguanine DNA alkyltransferase-mediated repair of O6-pyridyloxobutyl-2'-deoxyguanosine adducts in DNA.” Chem Res Toxicol. 24:1966-1975.
31. Mattern J, Eichhorn U., Kaina B, Volm M. (1998). "O6-methylguanine-DNA methyltransferase activity and sensitivity to cyclophosphamide and cisplatin in human lung tumor xenografts." Int J Cancer. 77: 919-922.
32. McElhinney RS, McMurry TB, Margison GP. (2003). “O6-alkylguanine-DNA alkyltransferase inactivation in cancer chemotherapy.” Mini Rev Med Chem. 3:471-485.
33. McManus FP, Fang Q, Booth JD, Noronha AM, Pegg AE, Wilds CJ. (2010). "Synthesis and characterization of an O6-2'-deoxyguanosine-alkyl-O6-2'-deoxyguanosine interstrand cross-link in a 5'-GNC motif and repair by human O6-alkylguanine-DNA alkyltransferase." Org Biomol Chem. 8: 4414-4426.
34. Mishina Y, Duguid EM, He C. (2006) “Direct reversal of DNA alkylation damage.” Chem Rev. 106:215-232.
35. Moore MH, Gulbis JM, Dodson EJ, Demple B, Moody PCE. (1994) “Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.” EMBO. 13: 1495-1501.
36. Pegg AE, Perry W, Bennett RA. (1981). “Partial hepatectomy increases the ability of rat liver extracts to catalyze removal of O6-methylguanine from alkylated DNA.” Biochem. J. 197:195-201.
37. Pegg AE. (2000). “Repair of O6-alkylguanine by alkyltransferases.” Mutat Res. 462:83-100.
38. Pegg AE, Goodtzova K, Loktionova NA, Kanugula S, Pauly GT, Moschel RC. (2001). “Inactivation of human O6-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O6-benzylguanine.” J Pharmacol Exp Ther. 296:958-965.
39. Pegg AE. (2011). “Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools.” Chem Res Toxicol. 24:618-639.
40. Philip PA, Souliotis VL, Harris AL, Salisbury A, Tates AD, Mitchell K, van Delft JH, Ganesan TS, Kyrtopoulos SA. (1996). “Methyl DNA adducts, DNA repair, and hypoxanthine-guanine phosphoribosyl transferase mutations in peripheral white blood cells from patients with malignant melanoma treated with dacarbazine and hydroxyurea.” Clin Cancer Res. 2:303-310.
41. Reagan MS, Pittenger C, Siede W, Friedberg EC. (1995) “Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene.” J Bacteriol. 177:364-371.
42. Schoonhoven NM, Murphy S., O'Flaherty DK, Noronha AM, Kornblatt MJ, Wilds CJ. (2008). "Synthesis, biophysical and repair studies of O6-2'-deoxyguanosine adducts by Escherichia coli OGT." Nucleic Acids Symp Ser (Oxf). 52: 449-450.
43. Scicchitano D, Jones RA, Kuzmich S, Gaffney B, Lasko DD, Essigmann JM, Pegg AE. (1986). “Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase.” Carcinogenesis. 7:1383-1386.
44. Srivenugopal KS, Yuan XH, Friedman HS, Ali-Osman F. (1996). “Ubiquitination-dependent proteolysis of O6-methylguanine-DNA methyltransferase in human and murine tumour cells following inactivation with O6-benzylguanine or 1,3-bis(2-chloroethyl)-1-nitrosurea.” Biochemistry 35: 1328-1334.
45. Takahashi M, Sakumi K, Sekiguchi M. (1990). “Interaction of Ada protein with DNA examined by fluorescence anisotropy of the protein.” ¬Biochemistry 29: 3431-3436.
46. Toorchen D, Topal MD. (1983). “Mechanisms of chemical mutagenesis and carcinogenesis: effects on DNA replication of methylation at the O6-guanine position of dGTP.” Carcinogenesis. 4:1591-1597.
47. Toorchen D, Lindamood C 3rd, Swenberg JA, Topal MD. (1984). “O6-methylguanine-DNA transmethylase converts O6-methylguanine thymine base pairs to guanine thymine base pairs in DNA.” Carcinogenesis. 5:1733-1735.
48. Tubbs JL, Pegg AE, Tainer JA. (2007). “DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy.” DNA Repair (Amst). 6:1100-1115.
49. Vaughan P, Lindahl T, Sedgwick B. (1993). “Induction of the adaptive response of Escherichia coli to alkylation damage by the environmental mutagen, methyl chloride.” Mutat Res. 293:249-257.
50. Verdemato PE, Brannigan JA, Damblon C, Zuccotto F, Moody PC, Lian LY. (2000). “DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase.” Nucleic Acids Res. 28:3710-3718.
51. Vora RA, Pegg AE, Ealick SE. (1998). “A new model for how O6-methylguanine-DNA methyltransferase binds DNA.” Proteins 32:3-6.
52. Wilkinson MC, Potter PM, Cawkwell L, Georgiadis P, Patel D, Swann PF, Margison GP. (1989). “Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.” Nucleic Acids Res. 17:8475-8484.
53. Wilson BD, Strauss M, Stickells BJ, Hoal-van Helden EG, van Helden P. (1994). “An assay for O6-alkylguanine-DNA alkyltransferase based on restriction endonuclease inhibition and magnetic bead separation of products.” Carcinogenesis. 15:2143-2148.
54. Xu-Welliver M, Pegg AE. (2002). “Degradation of the alkylated form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase.” Carcinogenesis. 23:823-830.
55. Zang H, Fang Q., Pegg AE, Guengerich FP. (2005). "Kinetic analysis of steps in the repair of damaged DNA by human O6-alkylguanine-DNA alkyltransferase." J Biol Chem. 280: 30873-30881.
56. Kozekov ID, Nechev LV, Moseley MS, Harris CM, Rizzo CJ, Stone MP, Harris TM. (2003). “DNA interchain cross-links formed by acrolein and crotonaldehyde.” J Am Chem Soc. 125:50-61.
57. Sancar A. (2003). “Structure and function of DNA photolyase and chryptochrome blue-light photoreceptors.” Chem Rev. 103:2203-2237.
58. Rydberg B, Lindahl T. (1982). “Non-enzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction.” EMBO J. 1:211-216.
59. Taverna P, Sedgwick B. (1996). “Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli.” J Bacteriol. 178:5105-5111.
60. Friedman JI, Jiang YL, Miller PS, Stivers JT. (2011). “Unique dynamic properties of DNA duplexes containing interstrand cross-links.” Biochemistry 50:882-890.
61. Jamieson ER, Lippard SJ. (1999). “Structure, Recognition, and Processing of Cisplatin-DNA Adducts.” Chem Rev. 99:2467-2498.
62. Wang D, Hara R, Singh G, Sancar A, Lippard SJ. (2003). “Nucleotide excision repair from site-specifically platinum-modified nucleosomes.” Biochemistry. 42:6747-6753.
63. Dooley PA, Tsarouhtsis D, Korbel GA, Nechev LV, Shearer J, Zegar IS, Harris CM, Stone MP, Harris TM. (2001). “Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link.” J Am Chem Soc. 123:1730-1739.
64. Dooley PA, Zhang M, Korbel GA, Nechev LV, Harris CM, Stone MP, Harris TM. (2003). “NMR determination of the conformation of a trimethylene interstrand cross-link in an oligodeoxynucleotide duplex containing a 5'-d(GpC) motif.” J Am Chem Soc. 125:62-72.
65. Guengerich FP. (2006). “Interactions of Carcinogen-Bound DNA with Individual DNA polymerases.” Chem Rev. 106:420-452.
66. Huang H, Kim HY, Kozekov ID, Cho YJ, Wang H, Kozekova A, Harris TM, Rizzo CJ, Stone MP. (2009). “Stereospecific formation of the (R)-γ-hydroxytrimethylene interstrand N2-dG: N2-dG cross-link arising from the γ -OH-1,N2-propano-2'-deoxyguanosine adduct in the 5'-CpG-3' DNA sequence.” J Am Chem Soc. 131:8416-8424.
67. Stevens K, Madder A. (2009). “Furan-modified oligonucleotides for fast, high-yielding and site-selective DNA inter-strand cross-linking with non-modified complements.” Nucleic Acids Res. 37:1555-1565.
68. Streeper RT, Cotter RJ, Colvin ME, Hilton J, Colvin OM. (1995). “Molecular pharmacology of hepsulfam, NSC 3296801: identification of alkylated nucleosides, alkylation site, and site of DNA cross-linking.” Cancer Res. 55:1491-1498.
69. Wilds CJ, Booth JD, Noronha AM. (2006). “Synthesis of oligonucleotides containing an O6-G-alkyl-O6-G interstrand crosslink.” Elsevier Tetrahedron Letters 47: 9125-9128.
70. Blanpain C, Mohrin M, Sotiropoulou PA, Passegué E. (2011). “DNA damage response in tissue-specific and cancer stem cells.” Cell Stem Cell. 8:16-29.
71. Burns JA, Dreij K, Cartularo L, Scicchitano DA. (2010). “O6-methylguanine induces altered proteins at the level of transcription in human cells.” Nucleic Acids Res. 38:8178-8187.
72. Nagel G, Brenner W, Johnsson K, Kaina B. (2003). “DNA repair protein O6-methylguanine-DNA methyltransferase in testis and testicular tumors as determined by a novel nonradioactive assay.” Anal Biochem. 321:38-43.
73. Teo AK, Oh HK, Ali RB, Li BF. (2001). “The modified human DNA repair enzyme O6-methylguanine-DNA methyltransferase is a negative regulator of estrogen receptor-mediated transcription upon alkylation DNA damage.” Mol Cell Biol. 21:7105-7114.
74. Bhavsar YP, Reilly SM, Wadkins RM. (2011). “Evaluation of fluorescent analogs of deoxycytidine for monitoring DNA transitions from duplex to functional structures.” J Nucleic Acids. 2011:986820.
75. Hardman SJ, Thompson KC. (2006). “Influence of base stacking and hydrogen bonding on the fluorescence of 2-aminopurine and pyrrolocytosine in nucleic acids.” Biochemistry 45:9145-9155.
76. Hudson RH, Choghamarani AG. (2007). “The 6-methoxymethyl derivative of pyrrolo-dC for selective fluorometric detection of guanosine-containing sequences.” Nucleosides, Nucleotides, Nucleic Acids 26:533-537.
77. Li T, Fu R, Park HG. (2010). “Pyrrolo-dC based fluorescent aptasensors for the molecular recognition of targets.” Chem Commun (Camb.) 46:3271-3273.
78. Martí AA, Jockusch S, Li Z, Ju J, Turro NJ. (2006). “Molecular beacons with intrinsically fluorescent nucleotides.” Nucleic Acids Res. 34:e50.
79. Ming X, Ding P, Leonard P, Budow S, Seela F. (2012) “Parallel-stranded DNA: enhancing duplex stability by the 'G-clamp' and a pyrrolo-dC derivative.” Org Biomol Chem. 10:1861-1869.
80. Moser AM, Patel M, Yoo H, Balis FM, Hawkins ME. (2000). “Real-time fluorescence assay for O6-alkylguanine-DNA alkyltransferase.” Anal Biochem. 281:216-222.
81. Park KS, Lee J
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