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Visualizing replication fork encounters with DNA interstrand crosslinks

  1. Author:
    James, Ryan C
    Bellani, Marina A
    Zhang, Jing
    Huang, Jing
    Shaik, Althaf
    Pokharel, Durga
    Gali,Himabindu
    Gichimu, Julia
    Thazhathveetil, Arun K
    Seidman, Michael M

  2. Author Address

    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States., Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States., Department of Neurosurgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China., Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, China., Horizon Discovery, Lafayette, CO, United States., Frederick National Laboratory for Cancer Research, Frederick, MD, United States., Nitto Denko Avecia Inc., Cincinnati, OH, United States., Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States. Electronic address: seidmanm@grc.nia.nih.gov.,
    1. Year: 2021
    2. Epub Date: 2021 Sept 21
  2. Journal: Methods in Enzymology
    1. 661
    2. Pages: 53-75
  4. Type of Article: Article
  1. Abstract:

    Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations. Copyright © 2021 Elsevier Inc. All rights reserved.

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External Sources

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  2. DOI: 10.1016/bs.mie.2021.08.015
  3. PMID: 34776223
  4. PII : S0076-6879(21)00358-X

Library Notes

  1. Fiscal Year: FY2020-2021
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