Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro.

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ø29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A(3) (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.

[1]  P. Nielsen.  Chemical and photochemical probing of DNA complexes , 1990, Journal of molecular recognition : JMR.

[2]  S. Mirkin,et al.  Triplex DNA structures. , 1995, Annual review of biochemistry.

[3]  M. Egholm,et al.  Sequence-specific transcription arrest by peptide nucleic acid bound to the DNA template strand. , 1994, Gene.

[4]  K. Hecker,et al.  Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases. , 1996, Biochemistry.

[5]  M. Egholm,et al.  Biochemical evidence that a D-loop is part of a four-stranded PNA-DNA bundle. Nickel-mediated cleavage of duplex DNA by a Gly-Gly-His bis-PNA. , 1996, Biochemistry.

[6]  G. Płucienniczak,et al.  Effect of DNA-interacting drugs on phage T7 RNA polymerase. , 1998, Acta biochimica Polonica.

[7]  J. Lown,,et al.  Recent developments in sequence selective minor groove DNA effectors. , 2000, Current medicinal chemistry.

[8]  R Gambari,et al.  Biospecific interaction analysis (BIA) of low-molecular weight DNA-binding drugs. , 2000, The Journal of pharmacology and experimental therapeutics.

[9]  R. Gambari,et al.  Polymerase-chain reaction as a tool for investigations on sequence-selectivity of DNA-drugs interactions. , 1994, Journal of biochemical and biophysical methods.

[10]  V. Potaman,et al.  Triple-Helical Nucleic Acids , 1995, Springer New York.

[11]  D. Phillips,et al.  Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs. , 1989, Biochemistry.

[12]  P. Nielsen,et al.  Electron microscopic studies of sequence‐specific recognition of duplex DNA by different ligands , 1994, Journal of molecular recognition : JMR.

[13]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[14]  J. Chaires,et al.  Sequence and structural selectivity of nucleic acid binding ligands. , 1999, Biochemistry.

[15]  S. Krawczyk,et al.  Triple helix formation inhibits transcription elongation in vitro. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Waring,et al.  Hydroxyl radical footprinting of the sequence‐selective binding of netropsin and distamycin to DNA , 1987, FEBS letters.

[17]  Robert W. Taylor,et al.  Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids , 1997, Nature Genetics.

[18]  M. Egholm,et al.  Peptide nucleic acids (PNAs): potential antisense and anti-gene agents. , 1993, Anti-cancer drug design.

[19]  E. Lukhtanov,et al.  Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  L. Blanco,et al.  ø29 DNA Polymerase Residue Ser122, a Single-stranded DNA Ligand for 3′-5′ Exonucleolysis, Is Required to Interact with the Terminal Protein* , 1998, The Journal of Biological Chemistry.

[21]  David Keller,et al.  Single-molecule studies of the effect of template tension on T7 DNA polymerase activity , 2000, Nature.

[22]  B. Wold,et al.  Inhibition of Klenow fragment DNA polymerase on double-helical templates by oligonucleotide-directed triple-helix formation. , 1994, Biochemistry.

[23]  P. Nielsen,et al.  Transcription-mediated binding of peptide nucleic acid (PNA) to double-stranded DNA: sequence-specific suicide transcription. , 1996, Nucleic acids research.

[24]  J. Goodisman,et al.  Quantitative footprinting analysis using a DNA-cleaving metalloporphyrin complex. , 1989, Biochemistry.

[25]  L. Hurley,et al.  Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase. , 1992, Biochemistry.

[26]  R. Hertzberg,et al.  Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage-inhibition patterns with methidiumpropyl-EDTA.Fe(II). , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[27]  R. Wells,et al.  Netropsin. A specific probe for A-T regions of duplex deoxyribonucleic acid. , 1974, The Journal of biological chemistry.

[28]  V. Croquette,et al.  Replication by a single DNA polymerase of a stretched single-stranded DNA. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  V V Demidov,et al.  An Artificial Primosome: Design, Function, and Applications , 2001, Chembiochem : a European journal of chemical biology.

[30]  Winter Ta.  Gene targeted agents: new opportunities for rational drug development. , 2000 .

[31]  T. Tullius,et al.  DNA footprinting with hydroxyl radical , 1988, Nature.

[32]  D. Boger,et al.  A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity. , 2001, Journal of the American Chemical Society.

[33]  T. Ellenberger,et al.  Tuning DNA “strings”: Modulating the rate of DNA replication with mechanical tension , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Crothers,et al.  High-resolution transcription assay for probing drug-DNA interactions at individual drug sites. , 2001, Methods in enzymology.

[35]  V V Demidov,et al.  PNA openers as a tool for direct quantification of specific targets in duplex DNA. , 1999, Journal of biomolecular structure & dynamics.

[36]  G. Verdine,et al.  High-resolution footprinting of sequence-specific protein–DNA contacts , 2002, Nature Biotechnology.

[37]  D. Crothers,et al.  Nucleic Acids: Structures, Properties, and Functions , 2000 .

[38]  M. Waring,et al.  Drug-nucleic acid interactions , 2001 .

[39]  G. Schroth,et al.  The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids. , 2001, Nucleic acids research.

[40]  K. L. Weiland,et al.  In vitro and in vivo DNA bonding by the CC-1065 analogue U-73975. , 1991, Biochemistry.