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Visualizing DNA Double Strand Break Repair with Single‐Molecule Microscopy
Author(s) -
Loparo Joseph,
Graham Thomas,
Walter Johannes
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.266.1
Subject(s) - dna ligase , dna repair , dna , non homologous end joining , dna repair protein xrcc4 , semiconservative replication , dna damage , biology , microbiology and biotechnology , computational biology , chemistry , biophysics , genetics , nucleotide excision repair , eukaryotic dna replication
DNA double strand breaks are an extremely toxic form of DNA damage and must be repaired to maintain genome stability. In human cells the majority of DNA double strand breaks are repaired by nonhomologous end joining (NHEJ), a versatile pathway capable of directly ligating even complicated DNA end structures. This flexibility comes with a price as the NHEJ machinery joins difficult ends in an error‐prone manner. How the NHEJ machinery assembles on DNA ends, holds the ends together and maximizes the fidelity of end joining remain open questions. In this talk I will describe how my laboratory is using single‐molecule imaging approaches in Xenopus laevis egg extracts to follow the repair of double strand DNA breaks in real time with nanometer resolution. We have found that DNA ends pass through at least two distinct synaptic states during repair. Initially ends are tethered in a long‐range synaptic state that requires Ku and DNA‐PKcs. The catalytic activity of DNA‐PKcs along with XLF and XRCC4‐LIG4 are then required to transition the DNA ends into close alignment prior to ligation. In order to understand the role of XLF and XRCC4 in this complex I will describe how we are fluorescently labeling these components and visualizing them during repair. Support or Funding Information This work was supported by a National Institutes of Health grant (R01GM115487) to J.J.L.