Visualizing the search for radiation-damaged DNA bases in real time

• Published in: Radiation physics and chemistry (Oxford, England : 1993) Vol. 128; pp. 126 - 133
• Main Authors: Lee, Andrea J., Wallace, Susan S.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.11.2016
• Subjects: Base excision repair; DNA glycosylases; Glycosylase diffusion; Search for radiation damage; Single molecule fluorescence; Wedge residue; Search for radiation damage; DNA glycosylases; Single molecule fluorescence; Wedge residue; Base excision repair; Glycosylase diffusion
• ISSN: 0969-806X; 1879-0895
• DOI: 10.1016/j.radphyschem.2016.05.011

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts. •TIRF microscopy provides insight into the DNA damage search by DNA glycosylases.•Glycosylases rotationally slide along the DNA backbone with a broad spectrum of diffusion constants.•Glycosylases diffuse randomly along DNA inserting a wedge amino acid into the duplex to locate damage.•Glycosylases stop upon encountering their substrate damage.•Glycosylases from different structural families exhibit the same search behavior.