Abstract
Modification of DNA nucleobases has a profound effect on genome function. We developed a method that maps the positions of the modified DNA nucleobases throughout genomic DNA. This method couples in vitro nucleobase excision with massively parallel DNA sequencing to determine the location of modified DNA nucleobases with single base precision. This protocol was used to map uracil incorporation and UV photodimers in DNA, and a modification of the protocol has been used to map sparse modification events in cells. The Excision-seq protocol is broadly applicable to a variety of base modifications for which an excision enzyme is available.
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References
Kumari S, Rastogi RP et al (2008) DNA damage: detection strategies. EXCLI J 7:44–62
Clark TA, Spittle KE, Turner SW et al (2011) Direct detection and sequencing of damaged DNA bases. Genome Integr 2:10
Baute J, Depicker A (2008) Base excision repair and its role in maintaining genome stability. Crit Rev Biochem Mol Biol 43:239–276
Hegde ML, Hazra TK, Mitra S (2008) Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells. Cell Res 18:27–47
Bryan DS, Ransom M, Adane B et al (2014) High resolution mapping of modified DNA nucleobases using excision repair enzymes. Genome Res 24:1534–1542
Sancar GB, Sancar A (2006) Purification and characterization of DNA photolyases. Methods Enzymol 408:121–156
Ryoji M, Katayama H, Fusamae H et al (1996) Repair of DNA damage in a mitochondrial lysate of Xenopus laevis oocytes. Nucleic Acids Res 24:4057–4062
Liu Z, Tan C, Guo X et al (2011) Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase. Proc Natl Acad Sci U S A 108:14831–14836
Li J, Liu Z, Tan C et al (2010) Dynamics and mechanism of repair of ultraviolet-induced (6-4) photoproduct by photolyase. Nature 466:887–890
Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Li H, Handsaker B, Wysoker A et al (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079
Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842
Karolchik D, Hinrichs AS, Kent WJ (2001) The UCSC Genome Browser. Wiley, Hoboken, NJ
Smith TS, Heger A, Sudbery I (2016) UMI-tools: modelling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. bioRxiv 051755
Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Holko M, Yefanov A, Lee H, Zhang N, Robertson CL, Serova N, Davis S, Soboleva A (2012) NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Research 41 (D1):D991–D995
Acknowledgments
We thank A. Sancar and D. Zhang for graciously providing purified photolyase enzymes during the development of the Excision-seq protocol. This work was supported in part by a March of Dimes Basil O’Connor Research Grant (J.H.), a Damon Runyon-Rachleff Innovation award (J.H.), a Research Scholar Grant from the American Cancer Society (RSG-13-216-01-DMC), and a Department of Defense Visionary Postdoc Award (to M.R., W81XWH-12-1-0333).
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Ransom, M., Bryan, D.S., Hesselberth, J.R. (2018). High-Resolution Mapping of Modified DNA Nucleobases Using Excision Repair Enzymes. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_6
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DOI: https://doi.org/10.1007/978-1-4939-7306-4_6
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