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The Nucleus pp 123-131 | Cite as

Purification of Proteins on Newly Synthesized DNA Using iPOND

  • Huzefa Dungrawala
  • David CortezEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1228)

Abstract

The replisome is a large protein machine containing multiple enzymatic activities needed to complete DNA replication. In addition to helicase and polymerases needed for copying the DNA, the replisome also contains proteins like DNA methyltransferases, histone chaperones, and chromatin modifying enzymes to couple DNA replication with chromatin deposition and establishment of the epigenetic code. In addition, since template DNA strands often contain DNA damage or other roadblocks to the replication machinery, replication stress response proteins associate with the replisome to stabilize, repair, and restart stalled replication forks. Hundreds of proteins are needed to accomplish these tasks. Identifying these proteins, monitoring their posttranslational modifications, and understanding how their activities are coordinated is essential to understand how the genome and epigenome are duplicated rapidly, completely, and accurately every cell division cycle. Here we describe an updated iPOND (isolation of proteins on nascent DNA) method to facilitate these analyses.

Key words

DNA replication Chromatin DNA repair iPOND EdU Click chemistry Replication stress PCNA DNA damage 

Notes

Acknowledgements

This work was supported by NCI grant R01CA136933 to D.C. We thank Bianca Sirbu and Frank Couch who contributed to the initial invention of iPOND.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiochemistryVanderbilt University School of MedicineNashvilleUSA

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