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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

Abstract

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:
Research Org.:
Institut de Recherches Scientifiques sur le Cancer, Villejuif (France)
OSTI Identifier:
6599643
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Virol.; (United States); Journal Volume: 62:11
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

Citation Formats

Mezzina, M., Menck, C.F.M., Courtin, P., and Sarasin, A.. Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands. United States: N. p., 1988. Web.
Mezzina, M., Menck, C.F.M., Courtin, P., & Sarasin, A.. Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands. United States.
Mezzina, M., Menck, C.F.M., Courtin, P., and Sarasin, A.. 1988. "Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands". United States. doi:.
@article{osti_6599643,
title = {Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands},
author = {Mezzina, M. and Menck, C.F.M. and Courtin, P. and Sarasin, A.},
abstractNote = {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 digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules.},
doi = {},
journal = {J. Virol.; (United States)},
number = ,
volume = 62:11,
place = {United States},
year = 1988,
month =
}
  • Several clones of simian virus 40 (SV40)-transformed hamster kidney cells, which are heterogeneous for induction of infectious SV40, have been studied. SV40 yields are low after induction with /sup 60/Co ..gamma.. irradiation or mitomycin C. In order to clarify the mechanism(s) by which virus is produced in induced cells, we analyzed the replication of viral DNA and production of virion (V) antigen and infectious virus after induction in various clones as well as in lytically infected permissive cells. Cells replicating SV40 DNA or synthesizing V antigen were visualized by in situ hybridization and immunofluorescence techniques, respectively. Only some cells inmore » induced cultures were found to produce SV40 and those which did were less efficient than lytically infected monkey cells. Mitomycin C or /sup 60/Co ..gamma.. irradiation acted by inducing more cells to replicate virus rather than by increasing the amount of SV40 released from individual cells. A greater proportion of cells could be induced to replicate SV40 DNA than to synthesize V antigen in all induced clones studied. Also, SV40 DNA replication was induced at lower doses of ..gamma.. irradiation than the production of either V antigen or infectious virus suggesting that synthesis of late virus protein is more restricted in induced cells than is replication of SV40 DNA. These findings indicate that one of the effects of induction treatments on SV40-transformed hamster cells is an enhancement of the cells' capacity to support SV40 replication.« less
  • The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT){sub 12} is also reported. To test the conclusions derived from these structures, residues identified as beingmore » involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.« less
  • Simian virus 40 minichromosomes, isolated from both virions (MV) and infected cells (MI), have highly compact structures in buffer containing 0.15 M NaCl and sediment with S values of about 90 to 100 and 115 to 130, respectively. Under the electron microscopy, also, MI appear the more compact of the two. Only 30 to 35% of the sites of origin and termination of replication in MV are freely available to the restriction endonucleases Bgl 1 and Bam H1. MV are similarly resistant to Eco R1 and Hpa II. In contrast, almost no sites in MI are available to any ofmore » the above single-cut endonucleases. In 0.6 M NaCl, MV and MI change to relaxed structures of 45 to 55 S and 50 to 60 S, respectively, containing 20 to 24 nucleosomes per genome, and become more sensitive to Bgl 1, Bam H1, Eco R1, and HPa II.« less
  • Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replicationmore » and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.« less