A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases

Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases β, η, ι and κ under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases ι and η. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.

[1]  David M. Wilson,et al.  Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1 , 2009, PloS one.

[2]  F. Sugawara,et al.  Penicilliols A and B, novel inhibitors specific to mammalian Y-family DNA polymerases. , 2009, Bioorganic & medicinal chemistry.

[3]  Sam Michael,et al.  A robotic platform for quantitative high-throughput screening. , 2008, Assay and drug development technologies.

[4]  Sam Michael,et al.  Compound Management for Quantitative High-Throughput Screening , 2008, JALA.

[5]  Ruili Huang,et al.  Fluorescence spectroscopic profiling of compound libraries. , 2008, Journal of medicinal chemistry.

[6]  V. Bohr,et al.  DNA damage, DNA repair, ageing and age-related disease , 2008, Mechanisms of Ageing and Development.

[7]  M. Yamada,et al.  Specificity of mutations induced by incorporation of oxidized dNTPs into DNA by human DNA polymerase eta. , 2008, DNA repair.

[8]  R. Woodgate,et al.  What a difference a decade makes: Insights into translesion DNA synthesis , 2007, Proceedings of the National Academy of Sciences.

[9]  E. G. Frank,et al.  Increased Catalytic Activity and Altered Fidelity of Human DNA Polymerase ι in the Presence of Manganese* , 2007, Journal of Biological Chemistry.

[10]  E. Crespan,et al.  8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins , 2007, Nature.

[11]  C. Gloeckner,et al.  Evolving a thermostable DNA polymerase that amplifies from highly damaged templates. , 2007, Angewandte Chemie.

[12]  Yan Xu,et al.  Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta. , 2007, Biochemistry.

[13]  R. Woodgate,et al.  Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. , 2007, Cancer research.

[14]  A. Marx,et al.  Evolving thermostable reverse transcriptase activity in a DNA polymerase scaffold. , 2006, Angewandte Chemie.

[15]  Kemin Wang,et al.  Real-time monitoring of DNA polymerase activity using molecular beacon. , 2006, Analytical biochemistry.

[16]  A. Lehmann,et al.  The Y-family DNA polymerase κ (pol κ) functions in mammalian nucleotide-excision repair , 2006, Nature Cell Biology.

[17]  S. West,et al.  Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination. , 2005, Molecular cell.

[18]  A. Marx,et al.  Enhanced fidelity in mismatch extension by DNA polymerase through directed combinatorial enzyme design. , 2005, Angewandte Chemie.

[19]  R. Burgess,et al.  Homogeneous fluorescent assay for RNA polymerase. , 2005, Analytical biochemistry.

[20]  A. Lehmann.  Replication of damaged DNA by translesion synthesis in human cells , 2005, FEBS letters.

[21]  Samuel H. Wilson,et al.  Identification of Small Molecule Synthetic Inhibitors of DNA Polymerase β by NMR Chemical Shift Mapping* , 2004, Journal of Biological Chemistry.

[22]  J. Sweasy,et al.  Is There a Link Between DNA Polymerase Beta and Cancer? , 2004, Cell cycle.

[23]  R. Woodgate,et al.  Snapshots of replication through an abasic lesion; structural basis for base substitutions and frameshifts. , 2004, Molecular cell.

[24]  Robert E. Johnson,et al.  Human DNA Polymerase ι Utilizes Different Nucleotide Incorporation Mechanisms Dependent upon the Template Base , 2004, Molecular and Cellular Biology.

[25]  Samuel H. Wilson,et al.  Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase ι by Controlled Proteolysis* , 2003, Journal of Biological Chemistry.

[26]  E. G. Frank,et al.  Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota. , 2002, Nucleic acids research.

[27]  Y. Shinkai,et al.  Polκ protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Fred Russell Kramer,et al.  Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. , 2002, Nucleic acids research.

[29]  A. Marx,et al.  A molecular beacon for quantitative monitoring of the DNA polymerase reaction in real-time. , 2002, Angewandte Chemie.

[30]  Yanbin Zhang,et al.  Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion. , 2002, DNA repair.

[31]  R. Woodgate,et al.  Unique misinsertion specificity of polι may decrease the mutagenic potential of deaminated cytosines , 2001, The EMBO journal.

[32]  T. Kunkel,et al.  The Y-family of DNA polymerases. , 2001, Molecular cell.

[33]  E. G. Frank,et al.  Altered nucleotide misinsertion fidelity associated with polι‐dependent replication at the end of a DNA template , 2001, The EMBO journal.

[34]  Toshiro Matsuda,et al.  Somatic mutation hotspots correlate with DNA polymerase η error spectrum , 2001, Nature Immunology.

[35]  S. Chaney,et al.  The Effect of DNA Structure on the Catalytic Efficiency and Fidelity of Human DNA Polymerase β on Templates with Platinum-DNA Adducts* , 2001, The Journal of Biological Chemistry.

[36]  E. G. Frank,et al.  Biochemical characterization of human DNA polymerase iota provides clues to its biological function. , 2001, Biochemical Society transactions.

[37]  Samuel H. Wilson,et al.  5'-Deoxyribose Phosphate Lyase Activity of Human DNA Polymerase ɩ in Vitro , 2001, Science.

[38]  Fenghua Yuan,et al.  Response of human DNA polymerase ι to DNA lesions , 2001 .

[39]  Yanbin Zhang,et al.  Error-prone lesion bypass by human DNA polymerase eta. , 2000, Nucleic acids research.

[40]  Fenghua Yuan,et al.  Error-free and error-prone lesion bypass by human DNA polymerase κ in vitro , 2000 .

[41]  J P McDonald,et al.  Misinsertion and bypass of thymine–thymine dimers by human DNA polymerase ι , 2000, The EMBO journal.

[42]  Satya Prakash,et al.  Eukaryotic polymerases ι and ζ act sequentially to bypass DNA lesions , 2000, Nature.

[43]  E. G. Frank,et al.  poliota, a remarkably error-prone human DNA polymerase. , 2000, Genes & development.

[44]  F. Hanaoka,et al.  Mechanisms of accurate translesion synthesis by human DNA polymerase η , 2000, The EMBO journal.

[45]  Satya Prakash,et al.  Fidelity of Human DNA Polymerase η* , 2000, The Journal of Biological Chemistry.

[46]  Robert E. Johnson,et al.  hRAD30 mutations in the variant form of xeroderma pigmentosum. , 1999, Science.

[47]  Chikahide Masutani,et al.  The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η , 1999, Nature.

[48]  Samuel H. Wilson,et al.  “Action-at-a-Distance” Mutagenesis , 1999, The Journal of Biological Chemistry.

[49]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[50]  S. H. Wilson,et al.  Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells. , 1998, Biochemistry.

[51]  S. Moncada,et al.  The Determination of Plasma Nitrite and Nitrate by Capillary Ion Analysis , 1995 .

[52]  A. Larsen,et al.  Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells. , 1995, Biochemical pharmacology.

[53]  K. Khrapko,et al.  An oligonucleotide hybridization approach to DNA sequencing , 1989, FEBS letters.

[54]  T. Schleich,et al.  Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein--nucleic acid interactions. , 1980, Biochemistry.

[55]  T. Blumenthal,et al.  The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid. , 1973, Biochemical and biophysical research communications.

[56]  H. Klenow,et al.  Effect of monovalent cations on the activity of the DNA polymerase of Escherichia coli B. , 1969, European journal of biochemistry.

[57]  M. Kimmel,et al.  Conflict of interest statement. None declared. , 2010 .

[58]  R. Shoemaker,et al.  Chemical library screen for novel inhibitors of Kaposi's sarcoma-associated herpesvirus processive DNA synthesis. , 2006, Antiviral research.

[59]  T. Kunkel,et al.  Functions of DNA polymerases. , 2004, Advances in protein chemistry.

[60]  P. Gearhart,et al.  DNA polymerase η is an A-T mutator in somatic hypermutation of immunoglobulin variable genes , 2001, Nature Immunology.

[61]  E. G. Frank,et al.  pol (cid:1) , a remarkably error-prone human DNA polymerase , 2000 .

[62]  C. Wilcox,et al.  Fluorometric Assay for DNA Polymerases and Reverse Transcriptase , 1999 .

[63]  H. Mukhtar,et al.  Polyphenols as cancer chemopreventive agents , 1995, Journal of cellular biochemistry. Supplement.

[64]  Samuel H. Wilson,et al.  [11] Purification and domain-mapping of mammalian DNA polymerase β , 1995 .

[65]  W. Beard,et al.  Purification and domain-mapping of mammalian DNA polymerase beta. , 1995, Methods in enzymology.

[66]  T. Tritton,et al.  Aurintricarboxylic acid is a nonspecific enzyme inhibitor. , 1976, Molecular pharmacology.

[67]  J. Peters,et al.  Action at a Distance , 2020 .