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Studying Topoisomerase 1-Mediated Damage at Genomic Ribonucleotides

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

Ribonucleotides incorporated into DNA by the DNA polymerases can be incised by Topoisomerase 1 (Top1) to initiate removal of ribonucleotides from the genome. This Top1-dependent ribonucleotide removal has been demonstrated to result in multiple forms of genome instability in yeast. Here, we describe both quantitative and qualitative assays to identify mutations and other forms of DNA damage resulting from Top1-cleavage at unrepaired genomic ribonucleotides.

Key words

DNA replication Ribonucleotide excision repair RNase H2 Topoisomerase 1 Replication stress Mutagenesis Genome instability 

Notes

Acknowledgments

We thank Mercedes Arana and Salahuddin Syed for thoughtful comments on the manuscript, and all Kunkel lab members for helpful discussions. This work was supported by Project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS).

Supplementary material

394256_1_En_17_MOESM1_ESM.pdf (158 kb)
Supplementary Excel File 1 Mutation rate calculation worksheet. A spreadsheet into which fluctuation analysis information and data can be entered into appropriate cells in order to determine median mutation rate (×10−7) and 95% Confidence Interval (CI) for a given strain. For the “Entry” sheet, values to be entered into each column are defined in row 1. Modifiable fields are highlighted in yellow and output values are highlight in green. f, mutant frequency is the mutant fraction. μ2, mutation rate is the probability of a mutation per cell per division. Both f and μ2 are calculated for each culture and a median μ2 value is then determined by sorting the values (XLS 497 kb)

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Genome Integrity and Structural Biology LaboratoryNational Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH)Research Triangle ParkUSA

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