Impact of DNA End Structure and Cellular Growth Phase on DNA Repair by Nonhomologous End‐Joining (NHEJ) in Saccharomyces cerevisiae

Abstract

DNA within the cell is constantly bombarded by endogenous sources of damage (replication errors, cellular metabolism) and exogenous sources (radiation, chemical exposure), resulting in the formation of more than 100,000 DNA lesions per cell daily. There are several potential consequences of this damage, including stimulation of a cell stress response, changes in gene transcription, induction of controlled cell death (apoptosis), and the activation of DNA repair pathways. Mutations causing impaired DNA repair are associated with multiple human genetic disorders, which frequently lead to an increased cancer risk. A double‐strand break (DSB) is the most deleterious type of DNA damage, as it promotes mutations, chromosomal rearrangements, and other genomic changes that can lead to cell death. The two primary pathways that repair DSBs are Homologous Recombination (HR) and Nonhomologous End‐joining (NHEJ). This project is focused on the NHEJ pathway in Saccharomyces cerevisiae, which is mediated by three main protein complexes: Yku70‐Yku80 (Yku), Mre11‐Rad50‐Xrs2 (Mrx), and Dnl4‐Lif1‐Nej1 (Dnl). Our experiments use plasmid NHEJ assays to measure the efficiency and accuracy of DSB repair in mutant strains of yeast deficient in DNA repair. This is done by transforming plasmids containing a DSB in one of two selectable markers into cells and utilizing the NHEJ pathway to repair the plasmid. The results are then quantified by measuring cellular growth on selective media to determine the degree of reduced repair efficiency. Preliminary results indicated that the Mrx DNA end‐bridging complex is situationally required for efficient NHEJ, while the Yku and Dnl complexes are always required. The requirement for Mrx was dependent on the cell growth phase, as it was lower in cultures with high levels of G1 cells. Mrx was always required for efficient repair of ends with 3′ single‐stranded overhangs, but not for DSBs with 5′ single‐stranded overhangs. Our hypothesis is that Mrx may play a protector role at DSB ends containing 3′ overhangs, shielding them from nucleases such as Exo1 or Sgs1‐Dna2 that may preferentially target this end structure. In addition, using an improved version of the plasmid assays, we have identified three new S. cerevisiae genes that affect NHEJ repair efficiency. Current experiments are investigating the role of chromatin remodeling proteins Arp5, Arp6, and Arp8, along with DNA end‐processing proteins, to assess their role in NHEJ repair.