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. (i) 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. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) 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. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.
Mezzina
M
M
Laboratory of Molecular Mutagenesis, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Menck
C F
CF
Courtin
P
P
Sarasin
A
A
D002499:Centrifugation, Density Gradient; D004260:DNA Repair; D004261:DNA Replication / Q000528:radiation effects*; D004277:DNA, Single-Stranded / Q000648:ultrastructure; D004279:DNA, Viral / Q000528:radiation effects* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000528:radiation effects; D008957:Models, Genetic; D011740:Pyrimidine Dimers / Q000032:analysis; D013539:Simian virus 40 / Q000235:genetics / Q000528:radiation effects*; D014466:Ultraviolet Rays; D014776:Virus Cultivation
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers