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. © 2012 Springer Science+Business Media New York.
CITATION STYLE
Nikolaishvili-Feinberg, N., & Cordeiro-Stone, M. (2012). Assays of bypass replication of genotoxic lesions in cell-free extracts. Methods in Molecular Biology, 920, 503–528. https://doi.org/10.1007/978-1-61779-998-3_34
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