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Methods for studying DNA double-strand break repair and its impact on genome integrity

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Methods for studying DNA double-strand break repair and its impact on genome integrity

Guest Editors
Jacob Paiano

Laboratory of Genome Integrity

Jacob Paiano is a Ph.D. candidate and NSF graduate fellow in the Immunology Graduate Group at the University of...

Dali Zong

Laboratory of Genome Integrity

Dr. Dali Zong is a senior scientist in the Laboratory of Genome Integrity headed by Dr. André Nussenzweig at the...

Collection Overview

Eukaryotic cells encounter thousands of DNA breaks each day as part of normal metabolic activity and proliferation. Among these, DNA double-strand breaks (DSBs) are particularly toxic; they not only impede critical cellular processes (e.g. DNA replication, transcription) but also facilitate mutations that can foster malignant transformation. The study of DSB induction and repair has traditionally relied heavily on the tracking of repair factor accumulation as cytologically visible foci (so-called ionizing radiation-induced foci or IRIF) at sites of DNA damage by immunofluorescence. While this assay is relatively simple to execute and can be easily adapted to a variety of cell line systems, it does not provide a measure for DSBs directly. Moreover, it is not possible to deduce solely from IRIF formation whether DSB repair would eventually take place (or the fidelity of such repair events). This collection will present a number of current approaches that allow accurate measurement of DSB repair and its mutagenic potential. These include (1) Comet assay; (2) cytogenetic analysis of metaphase spreads; (3) CRISPR-Cas9 initiated homologous recombination assay; and (4) ex vivo immunoglobulin class switch recombination assay. In addition, recent advances in cutting edge technologies which enable spatiotemporal dynamics of DNA replication (DNA combing) and DNA-protein interactions (DNA curtains) to be interrogated at the single-molecule level will be introduced. Together, the methods described in this collection are excellent complements to the traditional IRIF-based assay and will be useful for investigators studying all aspects of cellular DSB induction and repair.  



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