Development of New Assays for the Simultaneous Measurement of DNA Double‐Strand Break Repair by Multiple Pathways

Abstract

Cellular DNA is damaged by both endogenous and exogenous sources. Among the different types of DNA lesions that are formed, double‐strand breaks (DSBs) are considered to be the most consequential lesions. They can cause genomic instability and cellular death if not repaired via one of two pathways: nonhomologous end‐joining (NHEJ) or homologous recombination (HR). Deficiency of DSB repair is implicated in several congenital diseases and cancer‐prone disorders. In the budding yeast Saccharomyces cerevisiae NHEJ is accomplished by the actions of the Yku, Mrx, and Dnl4 protein complexes. Recent experiments in this lab have demonstrated that, in contrast to Yku and Dnl4, the Mrx complex is required for NHEJ repair of some DSB end structures but not others. The major goal of the current project is to characterize the role of Mrx and other protein complexes in repair by NHEJ and HR using newly developed plasmid and PCR fragment‐based assays. Experiments performed for the project demonstrated that repair of DSBs with 3′ single‐stranded DNA overhangs was reduced in yku70, mre11 and dnl4 mutants; however, the defect in NHEJ was strongly suppressed when EXO1, encoding a major 5′‐to‐3′ exonuclease, was inactivated in mre11 cells, i.e., in mre11 exo1 double mutants. By contrast, absence of the Sgs1‐Dna2 nuclease‐helicase complex (in mre11 sgs1 mutants) had no effect. NHEJ repair of DSBs with 5′ single‐stranded DNA overhangs was not changed by inactivation of EXO1 or SGS1. These findings implicate Mrx as a potential protector of DSB ends with 3′ overhangs during NHEJ repair and Exo1 as the main degradative nuclease. Additionally, we have developed new plasmid‐ and PCR fragment‐based repair assays that can analyze NHEJ and HR simultaneously using auxotrophic markers and type IIS restriction endonucleases. We anticipate that the new methods for simultaneous measurement will permit exploration of pathway choice in ways not previously possible.