title: Assays of bypass replication of genotoxic lesions in cell-free extracts.
abstract: The in vitro replication assay described here measures bidirectional replication of a circular double- stranded DNA template upon initiation at the SV40 origin. It models a single eukaryotic replication unit (replicon) and recapitulates the biochemical steps involved in the catalysis of both leading and lagging strand synthesis during semiconservative DNA replication. Except for the SV40 large T antigen, all other proteins necessary for initiation and assembly of functional replication forks are provided by the cell-free extract. This assay can be used to demonstrate bypass replication of genotoxic lesions. It supports replication across a specific damaged site on the template DNA (i.e., translesion synthesis) by specialized DNA polymerases. This chapter illustrates the efficient translesion synthesis of UV-induced thymine dimers by DNA polymerase eta.
authors.affiliation: Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center and Center for Environmental Health and Susceptibility, University of North Carolina at Chapel Hill, Chapel Hill, USA.
authors.forename: Nana
authors.initials: N
authors.lastname: Nikolaishvili-Feinberg
authors.forename: Marila
authors.initials: M
authors.lastname: Cordeiro-Stone
mesh_terms: D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004249:DNA Damage; D004261:DNA Replication; D004587:Electrophoresis, Agar Gel; D006367:HeLa Cells; D006801:Humans
chemical_list: D004247:DNA