Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae

Abstract

The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null (∆ or ∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4-dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 Thus, the quality of Ku's engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.

Nej1 Interacts with Mre11 to Regulate Tethering and Dna2 Binding at DNA Double-Strand Breaks

“…Here, we show that Nej1 recruitment to the break depends on the MRX complex and identify functions of Nej1 that are important for DNA repair pathway choice, including end-tethering and 5′ resection at the DSB. Nej1 was recently shown to be important for joining incompatible DNA ends (Yang et al, 2015), and our data support a model whereby Nej1 not only promotes canonical NHEJ but also might promote imprecise NHEJ under certain conditions (Emerson et al, 2018; Yang et al, 2015). In all, our work underscores the importance of Nej1 and the structural features of Rad50 in NHEJ.…”

A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger

“…These gene function studies were carried out nearly exclusively using NHEJ-deficient strains ( 24 , 36 ). A concern about using NHEJ-deficient strains is that it could have unexpected consequences in DNA repair capacity and promotes error-prone NHEJ ( 37 , 38 ). For example, a slight increase in the mutation rate for the A. flavus NRRL3357 Δ ku70 strain was reported, although no aberrant morphological changes were found ( 39 ).…”

A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus

“…Use of deficient-NHEJ repair pathway methods may generate unexpected effects in the reduction of DNA repair capacity (Emerson et al 2018;Pannunzio et al 2018).…”