Immunofluorescence-based methods to monitor DNA end resection.

Abstract

Double-strand breaks (DSBs) are the most deleterious among all types of DNA damage that can occur in the cell. These breaks arise from both endogenous (e.g., DNA replication stress) and exogenous insults (e.g., ionizing radiation). DSBs are principally repaired by one of two major pathways: nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an error-prone process that can occur in all phases of the cell cycle, while HR is limited to the S and G2 phases of the cell cycle when a sister chromatid is available as a template for error-free repair. The first step in HR is "DNA end resection," a process during which the broken DNA end is converted into a long stretch of 3'-ended single-stranded DNA (ssDNA). In recent years, DNA end resection has been identified as a pivotal step that controls "repair pathway choice," i.e., the appropriate choice between NHEJ and HR for DSB repair. Therefore, methods to quantitatively or semiquantitatively assess DNA end resection have gained importance in laboratories working on DNA repair. In this chapter, we describe two simple immunofluorescence-based techniques to monitor DNA end resection in mammalian cells. The first technique involves immuno-detection of replication protein A (RPA), an ssDNA-binding protein that binds to resected DNA. The second technique involves labeling of genomic DNA with 5-bromo-2'-deoxyuridine (BrdU) that can be detected by anti-BrdU antibody only after the DNA becomes single stranded due to resection. These methods are not complicated, do not involve sophisticated instrumentation or reporter constructs, and can be applied to most mammalian cell lines and, therefore, should be of broad utility as simple ways of monitoring DNA end resection in vivo.