Nucleic Acids Research, Vol 27, Issue 24 4734-4742, Copyright © 1999 by Oxford University Press
DH Oh and PC Hanawalt
The ability to target photochemical adducts to specific genomic DNA
sequences in cells is useful for studying DNA repair and mutagenesis in
intact cells, and also as a potential mode of gene-specific therapy. Triple
helix-forming DNA oligonucleotides linked to psoralen (psoTFOs) were
designed to deliver UVA-induced psoralen photoadducts to two distinct
sequences within the human interstitial collagenase gene. A primer
extension assay demonstrated that the appropriate psoTFO selectively
damages a collagenase cDNA target. Site-specific genomic psoTFO DNA adducts
were detected by a single-strand ligation PCR assay. The adduct, formed at
a single site by a psoTFO in purified genomic DNA, contrasted with the
multiple sites that were damaged within the observed segment of the
collagenase gene upon treatment with free psoralen and subsequent
photoactivation. When treated with psoTFOs, both repair-deficient
fibroblasts from xero- derma pigmentosum complementation group A and HT1080
fibrosarcoma cells exhibited site- specific DNA adducts following UVA
irradiation. Addition of phorbol ester, a transcriptional activator of the
collagenase gene, to xeroderma pigmentosum cells did not detectably alter
the initial levels of damage produced by psoTFOs, suggesting that further
stimulation of transcription neither improves accessibility of psoTFOs to
their targets nor enhances removal of non-covalently bound psoTFOs.
ARTICLES
Triple helix-forming oligonucleotides target psoralen adducts to specific chromosomal sequences in human cells
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA. doh@leland.stanford.edu
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