Abstract

Deoxyribonucleic acid (DNA) is constantly being damaged due to environmental chemicals, free radicals, and radiation. These damaging agents cause varying effects from phosphate, sugar, or base modification to double‐stranded breaks (DSBs) in the DNA. If DSBs are left untreated in a cell, the consequences can be grave, potentially causing loss of function, mutations, and cell death. In order to repair DSBs, two independent pathways are used, homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ is the preferred pathway in humans but secondary in the yeast Saccharomyces cerevisiae. The NHEJ pathway involves three major complexes in yeast cells: the Ku complex, Mrx complex, and DNA ligase IV complex. Each complex has a specific function in repairing DSBs.The Ku complex is a heterodimer found in both humans and yeast (Ku70 and Ku80 for humans, Yku70 and Yku80 in yeast), which is responsible for first binding to the broken ends of the DNA and protecting the ends from degradation. A mutation in the Ku70/Ku80 complex genes causes severe combined immunodeficiency (SCID) in humans and animals. The Mrx complex is composed of Mre11, Rad50, and Xrs2 in yeast and binds to the DNA ends to form an end‐bridging complex. The last complex is the DNA ligase IV complex that is composed of Dnl4 and Lif1. This complex performs the last step in NHEJ whose function is to ligate the two DNA ends together.Many past studies have studied NHEJ repair using plasmid end‐joining assays. In this method, cells are transformed with a plasmid containing a DSB that cannot be repaired by HR and the number of transformants with plasmids recircularized by NHEJ is determined. Previous reports observed that repair in yku, mrx and dnl4 mutants is reduced by 10–30 fold and suggested that the three major complexes are approximately equally important in the pathway. Using high efficiency transformation methods and early stationary phase cell cultures, we have been able to detect strong differences in the requirements for each complex. These results and additional experiments suggest that cell cycle phase distribution within cell cultures affects repair complex requirements.

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