6.8 Methoden Kapitel 3.4
6.8.2 Native Polyacrylamid-Gelelektrophorese
Die Wechselwirkungen von Proteinen mit DNA wurden mit Hilfe von nativer Polyacrylamid-Gelelektrophorese untersucht. Dafür wurden 7 % Polyacrylamid Gele hergestellt (in 1.5 mm Dicke). Die hergestellte Gellösung wurde zwischen zwei gereinigte Glasplatten (12 cm max.
Lauflänge) gegossen, und die entsprechenden Kämme für die Geltaschen eingesetzt. Die Proben wurden mit einem Sechstel Volumen Ladepuffer gemischt und auf das nicht denaturierende Gel aufgetragen. Als Längenreferenz wurden die Farbstoffe Bromphenolblau und Xylencyanol verwendet. Die Trennung der freien DNA von DNA-Protein-Komplexen
Methoden
erfolgte bei einer Spannung von 63 V für 5 min und 48 V für 4 h bei 4 °C. Die Detektion erfolgte autoradiographisch.
7 % natives Polyacrylamid Gel
Reagenz 100 ml
Acrylamid 29:1 23,2 ml
10x TBE 10 ml
Wasser 66,8 ml
APS 720 µl
TEMED 40 µl
Laufpuffer
Reagenz Endkonzentration
Tris- HCl 25 mM
Borsäure 25 mM
MgCl2 2 mM
EDTA 0.5 mM
6x Ladepuffer
Reagenz 100 ml
Glycerol 30 ml
Wasser 70 ml
Bromphenolblau 0.25 %
Xylencyanol 0.25 %
Literaturverzeichnis
7 Literaturverzeichnis
1. Watson, J. D. & Crick, F. H. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171, 737-8 (1953).
2. Brown, D. M. & Todd, A. R. Nucleic acids. Annu Rev Biochem 24, 311-38 (1955).
3. Khorana, H. G. Synthesis of nucleotides, nucleotide coenzymes and polynucleotides.
Fed Proc 19, 931-41 (1960).
4. Khorana, H. G. Total synthesis of a gene. Science 203, 614-25 (1979).
5. Matthaei, J. H. & Nirenberg, M. W. Characteristics and stabilization of DNAase-sensitive protein synthesis in E. coli extracts. Proc Natl Acad Sci U S A 47, 1580-8 (1961).
6. Nirenberg, M. W. & Matthaei, J. H. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A 47, 1588-602 (1961).
7. Zamecnik, P. C. & Stephenson, M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A 75, 280-4 (1978).
8. Brody, E. N. & Gold, L. Aptamers as therapeutic and diagnostic agents. J Biotechnol 74, 5-13 (2000).
9. Seitz, O. Chemische modifizierte Antisense-Oligonucleotide: neue Fortschritte auf dem Gebiet der Bindung von RNA und der Aktivierung der Ribonuclease H. Angew.
Chem. 111, 3674-3677 (1999).
10. Wilson, D. S. & Szostak, J. W. In vitro selection of functional nucleic acids. Annu Rev Biochem 68, 611-47 (1999).
11. Famulok, M. Oligonucleotide aptamers that recognize small molecules. Curr Opin Struct Biol 9, 324-9 (1999).
12. Famulok, M. & Mayer, G. Aptamers as tools in molecular biology and immunology.
Curr Top Microbiol Immunol 243, 123-36 (1999).
13. Wlotzka, B. et al. In vivo properties of an anti-GnRH Spiegelmer: an example of an oligonucleotide-based therapeutic substance class. Proc Natl Acad Sci U S A 99, 8898-902 (2002).
14. Leva, S. et al. GnRH binding RNA and DNA Spiegelmers: a novel approach toward GnRH antagonism. Chem Biol 9, 351-9 (2002).
15. Kool, E. T., Morales, J. C. & Guckian, K. M. Mimicking the Structure and Function of DNA: Insights into DNA Stability and Replication. Angew Chem Int Ed Engl 39, 990-1009 (2000).
16. Mao, C., Sun, W., Shen, Z. & Seeman, N. C. A nanomechanical device based on the B-Z transition of DNA. Nature 397, 144-6 (1999).
Literaturverzeichnis
17. Kornberg, A., Baker, TA. DNA Replication. Freeman, New York (1992).
18. Goodman, M. F. Error-prone repair DNA polymerases in prokaryotes and eukaryotes.
Annu Rev Biochem 71, 17-50 (2002).
19. Hubscher, U., Maga, G. & Spadari, S. Eukaryotic DNA polymerases. Annu Rev Biochem 71, 133-63 (2002).
20. Filée, J. F., P. Sen-Lin, Tang. Laurent, J. Evolution of DNA Polymerase Families:
Evidence for multiple gene exchange between cellular and viral proteins. J Mol Evol 54, 763-773 (2002).
21. Hubscher, U., Nasheuer, H. P. & Syvaoja, J. E. Eukaryotic DNA polymerases, a growing family. Trends Biochem Sci 25, 143-7 (2000).
22. Kunkel, T. A. & Bebenek, K. DNA replication fidelity. Annu Rev Biochem 69, 497-529 (2000).
23. Matsuda, T., Bebenek, K., Masutani, C., Hanaoka, F. & Kunkel, T. A. Low fidelity DNA synthesis by human DNA polymerase-eta. Nature 404, 1011-3 (2000).
24. Niimi, A. et al. Palm mutants in DNA polymerases alpha and eta alter DNA replication fidelity and translesion activity. Mol Cell Biol 24, 2734-46 (2004).
25. Li, Y. et al. Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7. Nat Struct Mol Biol 11, 784-90 (2004).
26. Marx, A. & Summerer, D. Molecular insights into error-prone DNA replication and error-free lesion bypass. Chembiochem 3, 405-7 (2002).
27. Mullis, K. B. Target amplification for DNA analysis by the polymerase chain reaction.
Ann Biol Clin (Paris) 48, 579-82 (1990).
28. Slatko, B. E. Thermal cycle dideoxy DNA sequencing. Mol Biotechnol 6, 311-22 (1996).
29. Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860-921 (2001).
30. Venter, J. C. et al. The sequence of the human genome. Science 291, 1304-51 (2001).
31. Tabor, S. & Richardson, C. C. A single residue in DNA polymerases of the
Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A 92, 6339-43 (1995).
32. Li, Y., Mitaxov, V. & Waksman, G. Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc Natl Acad Sci U S A 96, 9491-6 (1999).
33. Ling, M. M. & Robinson, B. H. Approaches to DNA mutagenesis: an overview. Anal Biochem 254, 157-78 (1997).
34. Joyce, C. Quantitative RT-PCR. A review of current methodologies. Methods Mol Biol
Literaturverzeichnis
35. Sninsky, J. J. The polymerase chain reaction (PCR): a valuable method for retroviral detection. Lymphology 23, 92-7 (1990).
36. Benecke, M. DNA typing in forensic medicine and in criminal investigations: a current survey. Naturwissenschaften 84, 181-8 (1997).
37. Syvanen, A. C. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet 2, 930-42 (2001).
38. Shi, M. M. Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem 47, 164-72 (2001).
39. Syvanen, A. C. From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 13, 1-10 (1999).
40. Pastinen, T., Kurg, A., Metspalu, A., Peltonen, L. & Syvanen, A. C. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 7, 606-14 (1997).
41. Ronaghi, M. Pyrosequencing sheds light on DNA sequencing. Genome Res 11, 3-11 (2001).
42. Myakishev, M. V., Khripin, Y., Hu, S. & Hamer, D. H. High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers.
Genome Res 11, 163-9 (2001).
43. Shi, M. M., Bleavins, M. R. & de la iglesia, F. A. Technologies for detecting genetic polymorphisms in pharmacogenomics. Mol Diagn 4, 343-51 (1999).
44. Cadwell, R. C. & Joyce, G. F. Mutagenic PCR. PCR Methods Appl 3, S136-40 (1994).
45. Miyazaki, C. et al. Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis. Protein Eng 12, 407-15 (1999).
46. Shibata, H., Kato, H. & Oda, J. Random mutagenesis on the Pseudomonas lipase activator protein, LipB: exploring amino acid residues required for its function. Protein Eng 11, 467-72 (1998).
47. Gardner, A. F., Joyce, C. M. & Jack, W. E. Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase. J Biol Chem 279, 11834-42 (2004).
48. Evans, S. J. et al. Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus. Nucleic Acids Res 28, 1059-66 (2000).
49. Knippers, R. Molekulare Genetik (Georg Thieme Verlag, 2006).
50. Benkovic, S. J., Valentine, A. M. & Salinas, F. Replisome-mediated DNA replication.
Annu Rev Biochem 70, 181-208 (2001).
51. Johnson, A. & O'Donnell, M. Cellular DNA replicases: components and dynamics at the replication fork. Annu Rev Biochem 74, 283-315 (2005).
52. Patel, P. H. & Loeb, L. A. DNA polymerase active site is highly mutable: evolutionary consequences. Proc Natl Acad Sci U S A 97, 5095-100 (2000).
Literaturverzeichnis
53. Jacobo-Molina, A. et al. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A 90, 6320-4 (1993).
54. Kim, Y. et al. Crystal structure of Thermus aquaticus DNA polymerase. Nature 376, 612-6 (1995).
55. Li, Y., Korolev, S. & Waksman, G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. Embo J 17, 7514-25 (1998).
56. Kiefer, J. R., Mao, C., Braman, J. C. & Beese, L. S. Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature 391, 304-7 (1998).
57. Doublie, S., Tabor, S., Long, A. M., Richardson, C. C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution. Nature 391, 251-8 (1998).
58. Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669-75 (1998).
59. Sarafianos, S. G. et al. Touching the heart of HIV-1 drug resistance: the fingers close down on the dNTP at the polymerase active site. Chem Biol 6, R137-46 (1999).
60. Summerer, D. & Marx, A. Differential minor groove interactions between DNA polymerase and sugar backbone of primer and template strands. J Am Chem Soc 124, 910-1 (2002).
61. Brautigam, C. A. & Steitz, T. A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr Opin Struct Biol 8, 54-63 (1998).
62. Patel, P. H., Suzuki, M., Adman, E., Shinkai, A. & Loeb, L. A. Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection. J Mol Biol 308, 823-37 (2001).
63. Steitz, T. A. DNA polymerases: structural diversity and common mechanisms. J Biol Chem 274, 17395-8 (1999).
64. Kiefer, J. R., Mao, C., Braman, J. C. & Beese, L. S. Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature 391, 304-307 (1998).
65. Kool, E. T. Replication of non-hydrogen bonded bases by DNA polymerases: a mechanism for steric matching. Biopolymers 48, 3-17 (1998).
66. Johnson, S. J., Taylor, J. S. & Beese, L. S. Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations.
Proc Natl Acad Sci U S A 100, 3895-900 (2003).
67. Li, Y. & Waksman, G. Crystal structures of a ddATP-, ddTTP-, ddCTP, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity. Protein Sci 10, 1225-33 (2001).
Literaturverzeichnis
68. Wang, J. et al. Crystal structure of a pol alpha family replication DNA polymerase from bacteriophage RB69. Cell 89, 1087-99 (1997).
69. Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. & Kraut, J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264, 1891-903 (1994).
70. Kool, E. T. Active site tightness and substrate fit in DNA replication. Annu Rev Biochem 71, 191-219 (2002).
71. Kool, E. T. Hydrogen bonding, base stacking, and steric effects in dna replication.
Annu Rev Biophys Biomol Struct 30, 1-22 (2001).
72. Moran, S., Ren, R.X.F., Rumney, S. & Kool, E.T. Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Journal of the American Chemical Society 119, 2056-2057 (1997).
73. Matsuda, S., Henry, A. A. & Romesberg, F. E. Optimization of unnatural base pair packing for polymerase recognition. J Am Chem Soc 128, 6369-75 (2006).
74. Tae, E. L., Wu, Y., Xia, G., Schultz, P. G. & Romesberg, F. E. Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. J Am Chem Soc 123, 7439-40 (2001).
75. Yu, C., Henry, A. A., Romesberg, F. E. & Schultz, P. G. Polymerase recognition of unnatural base pairs. Angew Chem Int Ed Engl 41, 3841-4 (2002).
76. Matsuda, S., Henry, A. A., Schultz, P. G. & Romesberg, F. E. The effect of minor-groove hydrogen-bond acceptors and donors on the stability and replication of four unnatural base pairs. J Am Chem Soc 125, 6134-9 (2003).
77. Spadari, S. et al. Lack of stereospecificity of some cellular and viral enzymes involved in the synthesis of deoxyribonucleotides and DNA: molecular basis for the antiviral activity of unnatural L-beta-nucleosides. Biochimie 77, 861-67 (1995).
78. Sosunov, V. V. et al. Stereochemical control of DNA biosynthesis. Nucleic Acids Res 28, 1170-5 (2000).
79. Asseline, U. et al. Synthesis and physicochemical properties of oligonucleotides built with either alpha-L or beta-L nucleotides units and covalently linked to an acridine derivative. Nucleic Acids Res 19, 4067-74 (1991).
80. Sniegowski, P. D., Gerrish, P. J., Johnson, T. & Shaver, A. The evolution of mutation rates: separating causes from consequences. Bioessays 22, 1057-66 (2000).
81. Brown, E. W., LeClerc, J. E., Kotewicz, M. L. & Cebula, T. A. Three R's of bacterial evolution: how replication, repair, and recombination frame the origin of species.
Environ Mol Mutagen 38, 248-60 (2001).
82. Powell, K. A. et al. Directed Evolution and Biocatalysis. Angew Chem Int Ed Engl 40, 3948-3959 (2001).
83. Sidhu, S. S. Phage display in pharmaceutical biotechnology. Curr Opin Biotechnol 11, 610-6 (2000).
Literaturverzeichnis
84. Ogino, H. & Ishikawa, H. Enzymes which are stable in the presence of organic solvents. J Biosci Bioeng 91, 109-16 (2001).
85. Matsuura, T. & Yomo, T. In vitro evolution of proteins. J Biosci Bioeng 101, 449-56 (2006).
86. Crameri, A., Raillard, S. A., Bermudez, E. & Stemmer, W. P. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288-91 (1998).
87. Stemmer, W. P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci U S A 91, 10747-51 (1994).
88. Shinkai, A., Patel, P. H. & Loeb, L. A. The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable. J Biol Chem 276, 18836-42 (2001).
89. Patel, P. H., Kawate, H., Adman, E., Ashbach, M. & Loeb, L. A. A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity. J Biol Chem 276, 5044-51 (2001).
90. Glick, E., Vigna, K. L. & Loeb, L. A. Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions. Embo J 20, 7303-12 (2001).
91. Lin, H., Cornish, V.W. Screening- und Selektionsmethoden für die Analyse von Proteinfunktionen in großem Maßstab. Angew Chem 114, 4580-4606 (2002).
92. Thum, O., Jager, S. & Famulok, M. Functionalized DNA: A New Replicable
Biopolymer We thank Dr. Andreas Marx, University of Bonn, for helpful advice and discussions. This work was supported by the Fonds der Chemischen Industrie, the Karl-Ziegler Stiftung, and the Deutsche Forschungsgemeinschaft. Angew Chem Int Ed Engl 40, 3990-3993 (2001).
93. Chaput, J. C. & Szostak, J. W. TNA synthesis by DNA polymerases. J Am Chem Soc 125, 9274-5 (2003).
94. Brakmann, S. & Nieckchen, P. The large fragment of Escherichia coli DNA
polymerase I can synthesize DNA exclusively from fluorescently labeled nucleotides.
Chembiochem 2, 773-7 (2001).
95. Patel, P. H. & Loeb, L. A. Getting a grip on how DNA polymerases function. Nat Struct Biol 8, 656-9 (2001).
96. Biles, B. D. & Connolly, B. A. Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR. Nucleic Acids Res 32, e176 (2004).
97. Garbesi, A. et al. L-DNAs as potential antimessenger oligonucleotides: a reassessment. Nucleic Acids Res 21, 4159-65 (1993).
98. Damha, M. J., Giannaris, P. A. & Marfey, P. Antisense L/D-oligodeoxynucleotide chimeras: nuclease stability, base-pairing properties, and activity at directing ribonuclease H. Biochemistry 33, 7877-85 (1994).
Literaturverzeichnis
99. Chang, C. N., Skalski, V., Zhou, J. H. & Cheng, Y. C. Biochemical pharmacology of (+)- and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents. J Biol Chem 267, 22414-20 (1992).
100. Skalski, V., Chang, C. N., Dutschman, G. & Cheng, Y. C. The biochemical basis for the differential anti-human immunodeficiency virus activity of two cis enantiomers of 2',3'-dideoxy-3'-thiacytidine. J Biol Chem 268, 23234-8 (1993).
101. Kavlick, M. F. et al. Genotypic and phenotypic characterization of HIV-1 isolated from patients receiving (--)-2',3'-dideoxy-3'-thiacytidine. Antiviral Res 28, 133-46 (1995).
102. Focher, F. et al. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. Nucleic Acids Res 23, 2840-7 (1995).
103. Semizarov, D. G. et al. Stereoisomers of deoxynucleoside 5'-triphosphates as substrates for template-dependent and -independent DNA polymerases. J Biol Chem 272, 9556-60 (1997).
104. Gardner, A. F. & Jack, W. E. Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases. Nucleic Acids Res 30, 605-13 (2002).
105. Woese, C. R., Kandler, O. & Wheelis, M. L. Towards a natural system of organisms:
proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A 87, 4576-9 (1990).
106. Southworth, M. W. et al. Cloning of thermostable DNA polymerases from
hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity. Proc Natl Acad Sci U S A 93, 5281-5 (1996).
107. Rodriguez, A. C., Park, H. W., Mao, C. & Beese, L. S. Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7. J Mol Biol 299, 447-62 (2000).
108. Ichida, J. K. et al. An in vitro selection system for TNA. J Am Chem Soc 127, 2802-3 (2005).
109. Ichida, J. K., Horhota, A., Zou, K., McLaughlin, L. W. & Szostak, J. W. High fidelity TNA synthesis by Therminator polymerase. Nucleic Acids Res 33, 5219-25 (2005).
110. Renders, M. et al. Enzymatic Synthesis of Phosphonomethyl Oligonucleotides by Therminator Polymerase. Angew Chem Int Ed Engl (2007).
111. Pavlov, A. R., Pavlova, N. V., Kozyavkin, S. A. & Slesarev, A. I. Recent developments in the optimization of thermostable DNA polymerases for efficient applications.
Trends Biotechnol 22, 253-60 (2004).
112. Lundberg, K. S. et al. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1-6 (1991).
113. Burgess, K. & Cook, D. Syntheses of nucleoside triphosphates. Chem Rev 100, 2047-60 (2000).
Literaturverzeichnis
114. Dabrowski, S. & Kur, J. Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei.
Protein Expr Purif 14, 131-8 (1998).
115. Brakmann, S. Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function. Cell Mol Life Sci 62, 2634-46 (2005).
116. Stemmer, W. P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389-91 (1994).
117. Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl 2, 28-33 (1992).
118. Fromant, M., Blanquet, S. & Plateau, P. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal Biochem 224, 347-53 (1995).
119. Meuer, S., Wittwer C., Nakagawara K. Rapid Cycle Real-Time PCR. Springer, Berlin (2001).
120. Morrison, T. B., Weis, J. J. & Wittwer, C. T. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24, 954-8, 960, 962 (1998).
121. DeVries, J. K. & Zubay, G. DNA-directed peptide synthesis. II. The synthesis of the alpha-fragment of the enzyme beta-galactosidase. Proc Natl Acad Sci U S A 57, 1010-2 (1967).
122. Tawfik, D. S. & Griffiths, A. D. Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16, 652-6 (1998).
123. Griffiths, A. D. & Tawfik, D. S. Directed evolution of an extremely fast
phosphotriesterase by in vitro compartmentalization. Embo J 22, 24-35 (2003).
124. Ghadessy, F. J., Ong, J. L. & Holliger, P. Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci U S A 98, 4552-7 (2001).
125. Lee, Y. F., Tawfik, D. S. & Griffiths, A. D. Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro
compartmentalisation. Nucleic Acids Res 30, 4937-44 (2002).
126. Griffiths, A. D. & Tawfik, D. S. Man-made enzymes--from design to in vitro compartmentalisation. Curr Opin Biotechnol 11, 338-53 (2000).
127. Lesley, S. A. Preparation and use of E. coli S-30 extracts. Methods Mol Biol 37, 265-78 (1995).
128. Lesley, S. A., Brow, M. A. & Burgess, R. R. Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies. J Biol Chem 266, 2632-8 (1991).
129. Woody, R. W. Methods in Enzymology (1995).
130. Baase, W. A. & Johnson, W. C., Jr. Secondary structure of nucleic acids in the folded
Literaturverzeichnis
131. Dussy, A. P. Dissertation. Universität Basel (1998).
132. Spence, R. A., Kati, W. M., Anderson, K. S. & Johnson, K. A. Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors. Science 267, 988-93 (1995).
133. Heidenreich, O., Kruhoffer, M., Grosse, F. & Eckstein, F. Inhibition of human immunodeficiency virus 1 reverse transcriptase by 3'-azidothymidine triphosphate.
Eur J Biochem 192, 621-5 (1990).
134. Balzarini, J., Baba, M., Pauwels, R., Herdewijn, P. & De Clerq, E. Anti-retrovirus activity of 3'-fluoro- and 3'-azido-substituted pyrimidine 2',3'-dideoxynucleoside analogues. Biochem Pharmacol 37, 2847-56 (1988).
135. Baba, M., Pauwels, R., Balzarini, J., Herdewijn, P. & De Clercq, E. Selective inhibition of human immunodeficiency virus (HIV) by 3'-azido-2', 3'-dideoxyguanosine in vitro.
Biochem Biophys Res Commun 145, 1080-6 (1987).
136. Summerer, D., Rudinger, N. Z., Detmer, I. & Marx, A. Enhanced fidelity in mismatch extension by DNA polymerase through directed combinatorial enzyme design. Angew Chem Int Ed Engl 44, 4712-5 (2005).
137. Joyce, C. M. & Steitz, T. A. Function and structure relationships in DNA polymerases.
Annu Rev Biochem 63, 777-822 (1994).
138. Steitz, T. A. The structural basis of the transition from initiation to elongation phases of transcription, as well as translocation and strand separation, by T7 RNA
polymerase. Curr Opin Struct Biol 14, 4-9 (2004).
139. Johnson, S. J. & Beese, L. S. Structures of mismatch replication errors observed in a DNA polymerase. Cell 116, 803-16 (2004).
140. Drew, H. R. et al. Structure of a B-DNA dodecamer: conformation and dynamics.
Proc Natl Acad Sci U S A 78, 2179-83 (1981).
141. Dickerson, R. E. et al. The anatomy of A-, B-, and Z-DNA. Science 216, 475-85 (1982).
142. Tereshko, V., Minasov, G., Egli, M. The Dickerson-Drew B-DNA dodecamer revisited at atomic resolution. Journal of the American Chemical Society 121, 6970-6970 (1999).
143. Detmer, I., Summerer, D. & Marx, A. DNA minor groove hydration probed with 4'-alkylated thymidines. Chem Commun (Camb), 2314-5 (2002).
144. Delarue, M., Poch, O., Tordo, N., Moras, D. & Argos, P. An attempt to unify the structure of polymerases. Protein Eng 3, 461-7 (1990).
145. Franklin, M. C., Wang, J. & Steitz, T. A. Structure of the replicating complex of a pol alpha family DNA polymerase. Cell 105, 657-67 (2001).
146. Astatke, M., Grindley, N. D. & Joyce, C. M. Deoxynucleoside triphosphate and pyrophosphate binding sites in the catalytically competent ternary complex for the polymerase reaction catalyzed by DNA polymerase I (Klenow fragment). J Biol Chem 270, 1945-54 (1995).
Literaturverzeichnis
147. Rechkoblit, O., Amin, S. & Geacintov, N. E. Primer length dependence of binding of DNA polymerase I Klenow fragment to template-primer complexes containing site-specific bulky lesions. Biochemistry 38, 11834-43 (1999).
148. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799-816 (2007).
149. Boosalis, M. S., Petruska, J. & Goodman, M. F. DNA polymerase insertion fidelity.
Gel assay for site-specific kinetics. J Biol Chem 262, 14689-96 (1987).
150. Creighton, S., Bloom, L. B. & Goodman, M. F. Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymol 262, 232-56 (1995).
Anhang