Abstract
A rapid, convenient and safe in vitro assay system for base excision repair is described. Whole cell extracts are prepared by detergent-based cell lysis and provide a vigorous activity of AP site repair. A circular DNA substrate is used for detection of both DNA polymerase β-dependent and proliferating cell nuclear antigen (PCNA)-dependent pathways. Repaired and unrepaired DNA substrates are separated by agarose gel electrophoresis as a linear DNA molecule and a nicked circular molecule, respectively, and detected by staining with SYBR Green I. This assay system does not require radioactive substrates or nucleotides, and provides a sensitivity in which 10 ng of a DNA substrate per reaction is sufficient for quantitative repair analysis.
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Instruction manual for Bio-Rad Protein Assay.
Acknowledgments
The author thanks C. C. Stobbe for critical reading of the manuscript. This work is supported by National Institutes of Health Grants CA06927, CA63154, and an appropriation from the Commonwealth of Pennsylvania.
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© 2006 Humana Press Inc.
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Matsumoto, Y. (2006). Base Excision Repair in Mammalian Cells. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:365
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DOI: https://doi.org/10.1385/1-59259-973-7:365
Publisher Name: Humana Press
Print ISBN: 978-1-58829-513-2
Online ISBN: 978-1-59259-973-8
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