Bypass replication in vitro of UV-induced photoproducts blocking leading or lagging strand synthesis.

Abstract

In vitro replication assays were used to determine the capacity of HeLa extracts to replicate past one of the two major photoproducts produced by ultraviolet radiation at adjacent thymines in duplex DNA, namely, the cis,syn cyclobutane dimer ([c,s]TT) and the 6-4 pyrimidine-pyrimidone adduct ([6-4]TT). The site-specific photoproduct was placed on the template either to the leading strand or to the lagging strand of nascent DNA with respect to the first fork encountering the lesion during bidirectional replication of closed circular duplex molecules carrying the SV40 origin. Replication products from time-course reactions were fractionated by gel electrophoresis in the presence of ethidium bromide. Recognition and quantification of true translesion synthesis products, i.e., newly synthesized closed circular molecules carrying the photoproduct, were aided by specific substrate modifications (a T:T mismatch in a unique PstI site nearby the photoproduct) and improved assay conditions (internal standard to control for completion of PstI digestion). Extracts from HeLa cells, which express DNA polymerase eta, were competent to replicate past the [c,s]TT on either strand. The efficiency of bypass replication of the [c,s]TT on the template to the leading or the lagging strand was 71% and 67%, respectively. The same extracts demonstrated very low efficiency of translesion synthesis (at most 8-10%) of the [6-4]TT on either template position. Replication-competent cell-free extracts from other human cells were also deficient in the bypass of the [6-4]TT in vitro.