Methods for Studying DNA Double-strand Break Repair and its Impact on Genome Integrity

TOPICAL COLLECTIONS

Guest Editors

Jacob Paiano

University of Pennsylvania, National Institutes of Health Immunology Graduate Group, National Cancer Institute, Center for Cancer Research, Laboratory of Genome Integrity

Jacob Paiano is a PhD candidate and NSF graduate fellow in the Immunology Graduate Group at the University of Pennsylvania. He received his BA in biology from Case Western Reserve University in 2012. Afterward, as a research assistant at Case, he uncovered roles for the local production and signaling of complement proteins in B cell activation and autoimmunity. He is currently working on his graduate thesis in the lab of André Nussenzweig in the National Cancer Institute at NIH studying cell cycle-regulated DNA damage and repair. Specifically, he is elucidating mechanisms of DNA break processing and recombination in mammalian meiosis and mitotic pathways of DNA replication in stressed cells.

Dali Zong

National Institutes of Health, National Cancer Institute, Center for Cancer Research, Laboratory of Genome Integrity

Dr. Dali Zong is a senior scientist in the Laboratory of Genome Integrity headed by Dr. André Nussenzweig at the National Cancer Institute in Bethesda, Maryland. The primary focus of his research is to elucidate how dysregulation of cellular DNA double-strand break repair pathways drive tumorigenesis and identify therapeutic opportunities to target vulnerabilities specific to repair-deficient cells. His recent work has led to the breakthrough discovery that a heterozygous mutation in the breast/ovarian cancer susceptibility gene BRCA1 can force cells to rely on an alternative repair pathway to preserve genome stability. He is an expert in various biochemical, cell biological and genetics/genomics tools, as well as diverse molecular imaging techniques. Prior to joining the NIH, Dr. Zong completed his Ph.D. training at the Karolinska Institutet in Stockholm, Sweden, where he also received his MSc in Biomedical Sciences.

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.