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Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
Published on: August 6, 2012
Assays of bypass replication of genotoxic lesions in cell-free extracts
Nana Nikolaishvili-Feinberg1, Marila Cordeiro-Stone
1Department 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.
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.
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