Abstract
We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki fragments. We delineate a method to extract, end label, and visualize Okazaki fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects of DNA replication and chromatin assembly.
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Acknowledgment
This work was supported by NIH grant GM102253 to I.W.
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Smith, D.J., Yadav, T., Whitehouse, I. (2015). Detection and Sequencing of Okazaki Fragments in S. cerevisiae . In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_10
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DOI: https://doi.org/10.1007/978-1-4939-2596-4_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2595-7
Online ISBN: 978-1-4939-2596-4
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