Journal Article Aphidicolin and 1-β-D-arabinofuranosylcytosine strongly inhibit transcriptionally active DNA repair in normal human fibroblasts Get access Razmik Mirzayans, Razmik Mirzayans 3 1Departments of Oncology, University of AlbertaEdmonton, Alberta T6G 2Z3, Canada 3To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Kevin Dietrich, Kevin Dietrich Search for other works by this author on: Oxford Academic PubMed Google Scholar Malcolm C. Paterson Malcolm C. Paterson 1Departments of Oncology, University of AlbertaEdmonton, Alberta T6G 2Z3, Canada2Biochemistry, University of AlbertaEdmonton, Alberta T6G 2Z3, Canada Search for other works by this author on: Oxford Academic PubMed Google Scholar Carcinogenesis, Volume 14, Issue 12, December 1993, Pages 2621–2626, https://doi.org/10.1093/carcin/14.12.2621 Published: 01 December 1993 Article history Received: 22 March 1993 Revision received: 09 September 1993 Accepted: 20 September 1993 Published: 01 December 1993
Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53 function. In this study we have examined the ability of these p53-deficient strains to carry out the long-patch, mode of excision repair, mediated by DNA polymerases δ and ɛ after exposure to 60Co γ radiation or far ultraviolet (UV) (chiefly 254 nm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (ape) or 1-β-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting nonligated strand incision events) by alkaline sucrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both γ rays and UV light. The repair anomaly in UV-irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both apc and araC decreased the level of UV-induced UDS by ∼75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xerodermapigentosum and Cockayne's syndrome. Together these results imply a critical role for wild-type p53 protein in DNA polymerase δ/ɛ-mediated excision repair, both the mechanism operating on the entire genome and that acting onexpressed genes.
The quantitative appraisal of the number of foreign gene copies integrated within the genomes of stably transfected cells is most conveniently performed using the strategy known as Southern blotting (1,2). First introduced by E. M. Southern in 1975 (2) the basic protocol involves the following steps: Size-fractionated DNA is first transferred from a gel matrix to a solid support under conditions that prevent self-annealing. The DNA molecules are then immobilized (covalently linked to the support) and processed for hybridization to a radiolabeled probe that has a nucleotide sequence complementing that of the target sequence to be detected. The blot is then washed extensively to remove unreacted probe molecules, and the hybrids formed between the probe and target sequences are revealed by autoradiography. The relative intensity of each autoradiographic signal will reflect the amount of hybridized material present. Quantification may be performed visually, by scanning laser densitometry, or, if the hybrids are sufficiently radioactive, by liquid scintillation counting.
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