UV lesions located on the leading strand inhibit DNA replication but do nor inhibit SV40 T-antigen helicase activity

DNA replication in eucaryotic cells involves a variety of proteins which sy nthesize the leading and lagging strands in an asymmetric coordinated manne r. 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 D NA replication-competent extracts made from human HeLa cells. Two dimension al agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand templa te. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesio n, we tested the effect of these single photoproducts on the unwinding acti vity 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 in hibited by UV-induced DNA lesions in double-stranded DNA substrate. (C) 200 0 Elsevier Science B.V. All lights reserved.