UV lesions located on the leading strand inhibit DNA replication but do not inhibit SV40 T-antigen helicase activity.
DNA replication in eucaryotic cells involves a variety of proteins which synthesize the leading and lagging strands in an asymmetric coordinated manner. To analyse the effect of this asymmetry on the translesion synthesis of UV-induced lesions, we have incubated SV40 origin-containing plasmids with a unique site-specific cis, syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct on either the leading or lagging strand template with DNA replication-competent extracts made from human HeLa cells. Two dimensional agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand template. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesion, we tested the effect of these single photoproducts on the unwinding activity of the SV40 T antigen, the major helicase in our in vitro replication assay. We showed that the activity of the SV40 T-antigen helicase is not inhibited by UV-induced DNA lesions in double-stranded DNA substrate.
Veaute
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Institut de Recherches sur le Cancer, UPR 42 CNRS, Laboratoire de Génétique Moléculaire, BP 8, 7, rue Guy-Moquet, 94801, Villejuif, France.
Mari-Giglia
G
G
Lawrence
C W
CW
Sarasin
A
A
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D002457:Cell Extracts; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D011740:Pyrimidine Dimers / Q000378:metabolism; D011744:Pyrimidinones / Q000378:metabolism; D014466:Ultraviolet Rays
D000952:Antigens, Polyomavirus Transforming; D002457:Cell Extracts; D011740:Pyrimidine Dimers; D011744:Pyrimidinones; D004247:DNA; D004265:DNA Helicases