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Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

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Acknowledgment

We thank Dr. Albert Serra Cardona for editing this protocol. This work was supported by NIH grants GM118015 to Z.Z and C.Y. is supported by the Edward C. Kendall Fellowship.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA

    Chuanhe Yu Ph.D.

  2. Institute for Cancer Genetics, Columbia University, 1130 St. Nicholas Avenue, New York, NY, USA

    Haiyun Gan Ph.D. & Zhiguo Zhang Ph.D.

  3. Department of Pediatrics, Columbia University, 1130 St. Nicholas Avenue, New York, NY, USA

    Haiyun Gan Ph.D. & Zhiguo Zhang Ph.D.

  4. Department of Genetics and Development, Columbia University, 1130 St. Nicholas Avenue, New York, NY, USA

    Haiyun Gan Ph.D. & Zhiguo Zhang Ph.D.

  5. Irving Cancer Research Center, ICRC 407A, New York, NY, 10032, USA

    Haiyun Gan Ph.D. & Zhiguo Zhang Ph.D.

Authors
  1. Chuanhe Yu Ph.D.
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  2. Haiyun Gan Ph.D.
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  3. Zhiguo Zhang Ph.D.
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Corresponding author

Correspondence to Zhiguo Zhang Ph.D. .

Editor information

Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Yu, C., Gan, H., Zhang, Z. (2018). Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast. 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_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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