TARGETED MUTAGENESIS OF DNA VIA TRIPLE HELIX FORMATION

Summary

Principal Investigator: Peter M Glazer
Affiliation: Yale University
Country: USA
Abstract: The ability to selectively mutate and inactivate specific genes in mammalian cells would be a valuable research tool to probe critical pathways associated with cancer and other diseases and might eventually have therapeutic applications. Oligonucleotides can bind to duplex DNA and form triple helices in a sequence- specific manner. Work funded by this grant has shown that by linking a triple helix-forming oligonucleotide (TFO) to a mutagen, the sequence specificity of triplex formation can be imparted to the action of the mutagen, and so DNA damage and thereby mutations can be directed to a specific site. TFOs conjugated to psoralen were found to produce site-specific mutations in supF reporter genes in an SV40 vector in cells, at frequencies up to 2 percent. It was also found that triplex formation, even in the absence of a tethered mutagen, can be mutagenic and that triplexes can both stimulate and inhibit cellular repair pathways. Recently, targeted mutagenesis of chromosomal genes was observed, using either DNA TFOs or oligomers composed of peptide nucleic acids (PNAs), with successful targeting of a supF gene in a recoverable, chromosomally integrated lambda vector and of the hprt gene. In this renewal application, work is proposed to further investigate chromosomal gene targeting by TFOs, using new lambda vectors containing modified supF reporter genes optimized for studying triplex-directed mutagenesis. Experiments will focus on testing the influence of triplex motif, target site transcription, cell cycle phase, and chromatin structure on chromosome accessibility and TFO-mediated targeting. A series of novel TFO modifications, including base, sugar, and backbone changes, will be provided by a team of collaborators assembled by the PI and will be tested for their effect on triplex formation, stability, and mutagenesis to identify optimal reagents for gene targeting in cells. Additional experiments will further test chromosome targeting by PNAs and will explore the utility of psoralen-PNA conjugates. The roles of DNA damage and repair in the triplex-mediated mutagenesis will be examined, to identify effective reagents for genome modification and to probe cellular pathways of triple helix DNA metabolism. A series of novel TFO- mutagen conjugates will be tested, including alkylating agents, camptothecin, and triplex-specific intercalators. The repair of triplex-associated DNA damage and of triplexes, themselves, will be investigated in human cell extracts, with an emphasis on assays to examine specific aspects of the nucleotide excision repair (NER) pathway, including repair endonuclease incisions, excision of damaged fragments, and repair-associated DNA synthesis.
Funding Period: 1995-07-01 - 2004-02-29
more information: NIH RePORT

Top Publications

  1. ncbi Distance and affinity dependence of triplex-induced recombination
    Melissa P Knauert
    Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, Connecticut 06520, USA
    Biochemistry 44:3856-64. 2005
  2. ncbi Triplex-induced recombination and repair in the pyrimidine motif
    Jennifer M Kalish
    Department of Therapeutic Radiology, Yale University School of Medicine PO Box 208040, HRT 140, New Haven, CT 06520-8040, USA
    Nucleic Acids Res 33:3492-502. 2005
  3. ncbi Triplex-stimulated intermolecular recombination at a single-copy genomic target
    Melissa P Knauert
    Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8040, USA
    Mol Ther 14:392-400. 2006
  4. ncbi Repair and recombination induced by triple helix DNA
    Joanna Y Chin
    Department of Genetics, Yale University School of Medicine, New Haven, CT 06520 8040, USA
    Front Biosci 12:4288-97. 2007
  5. ncbi Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids
    Ki Hyun Kim
    Department of Therapeutic Radiology, Yale University School of Medicine, P O Box 208040, New Haven, CT 06520 8040, USA
    Nucleic Acids Res 35:7604-13. 2007
  6. ncbi Triplex-mediated gene modification
    Erica B Schleifman
    Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
    Methods Mol Biol 435:175-90. 2008
  7. ncbi Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids
    Joanna Y Chin
    Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
    Proc Natl Acad Sci U S A 105:13514-9. 2008
  8. ncbi Repair of DNA lesions associated with triplex-forming oligonucleotides
    Joanna Y Chin
    Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, 15 York Street, New Haven, CT 06510, USA
    Mol Carcinog 48:389-99. 2009
  9. ncbi Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids
    Pallavi Lonkar
    Department of Therapeutic Radiology, Yale University School of Medicine, P O Box 208040, New Haven, CT 06520 8040, USA
    Nucleic Acids Res 37:3635-44. 2009

Detail Information

Publications9

  1. ncbi Distance and affinity dependence of triplex-induced recombination
    Melissa P Knauert
    Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, Connecticut 06520, USA
    Biochemistry 44:3856-64. 2005
    ..Together, these results indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation...
  2. ncbi Triplex-induced recombination and repair in the pyrimidine motif
    Jennifer M Kalish
    Department of Therapeutic Radiology, Yale University School of Medicine PO Box 208040, HRT 140, New Haven, CT 06520-8040, USA
    Nucleic Acids Res 33:3492-502. 2005
    ..By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting...
  3. ncbi Triplex-stimulated intermolecular recombination at a single-copy genomic target
    Melissa P Knauert
    Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8040, USA
    Mol Ther 14:392-400. 2006
    ..These results provide further support for the development of TFOs as reagents to stimulate site-specific correction of defective human genes...
  4. ncbi Repair and recombination induced by triple helix DNA
    Joanna Y Chin
    Department of Genetics, Yale University School of Medicine, New Haven, CT 06520 8040, USA
    Front Biosci 12:4288-97. 2007
    ..This review surveys the biological applications of TFOs, with particular attention to their recombinogenic and mutagenic potential, and summarizes available evidence for the mechanism of triplex and triplex-associated repair...
  5. ncbi Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids
    Ki Hyun Kim
    Department of Therapeutic Radiology, Yale University School of Medicine, P O Box 208040, New Haven, CT 06520 8040, USA
    Nucleic Acids Res 35:7604-13. 2007
    ..These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules...
  6. ncbi Triplex-mediated gene modification
    Erica B Schleifman
    Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
    Methods Mol Biol 435:175-90. 2008
    ..Target site identification, binding molecule design, as well as various methods to test binding and assess gene modification are described...
  7. ncbi Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids
    Joanna Y Chin
    Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
    Proc Natl Acad Sci U S A 105:13514-9. 2008
    ..This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells...
  8. ncbi Repair of DNA lesions associated with triplex-forming oligonucleotides
    Joanna Y Chin
    Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, 15 York Street, New Haven, CT 06510, USA
    Mol Carcinog 48:389-99. 2009
    ..This review examines the evidence for DNA repair of triplex-associated lesions...
  9. ncbi Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids
    Pallavi Lonkar
    Department of Therapeutic Radiology, Yale University School of Medicine, P O Box 208040, New Haven, CT 06520 8040, USA
    Nucleic Acids Res 37:3635-44. 2009
    ..These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction...