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Title: Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands

The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Carins-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestionmore » causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules.« less
Authors:
; ; ;
Publication Date:
OSTI Identifier:
6599643
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Virol.; (United States); Journal Volume: 62:11
Research Org:
Institut de Recherches Scientifiques sur le Cancer, Villejuif (France)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; ANIMAL CELLS; DNA REPLICATION; ELECTRON MICROSCOPY; GENETIC RADIATION EFFECTS; MAN; MOLECULAR STRUCTURE; MONKEYS; NUCLEASES; SIMIAN VIRUS; ULTRAVIOLET RADIATION; XERODERMA PIGMENTOSUM; ANIMALS; BIOLOGICAL EFFECTS; BIOLOGICAL RADIATION EFFECTS; DISEASES; ELECTROMAGNETIC RADIATION; ENZYMES; ESTERASES; GENETIC EFFECTS; HYDROLASES; MAMMALS; MICROORGANISMS; MICROSCOPY; NUCLEIC ACID REPLICATION; PARASITES; PHOSPHODIESTERASES; PRIMATES; RADIATION EFFECTS; RADIATIONS; SKIN DISEASES; VERTEBRATES; VIRUSES 560120* -- Radiation Effects on Biochemicals, Cells, & Tissue Culture