yKu70-yKu80 overexpression suppresses GCR formation induced by different types of DNA damage or generated spontaneously. The increased GCR frequency was suppressed by the overexpression of the yKu70-yKu80 heterodimer in the presence of 0.1% MMS (A), 0.5 μg/ml bleomycin (Bleo) (B), and a single DSB introduced by an HO endonuclease (C). (D) Mcm1, a DNA binding protein, did not suppress the GCRs induced by MMS treatment. (E) GCR rates in different GCR mutator strains were reduced by Ku overexpression. Each GCR rate of a GCR mutator strain with control plasmids was recalculated to 100%, and the GCR rate with Ku overexpression was converted to a percentage compared with that of strains carrying control plasmids. 100% GCR rate presented here for rfa1-t33, mre11, rad27, and rfa1-t33 rad24 are 5.8 × 10⁻⁷, 5.9 × 10⁻⁷, 2.2 × 10⁻⁶, and 4.9 × 10⁻⁷, respectively. “+” and “−” represent the presence and absence of indicated proteins or the treatment and no treatment of indicated DNA damaging agents, respectively.

GCR suppression by Ku overexpression after MMS treatment is mediated by its crosstalk with cell cycle checkpoints. (A) Ku overexpression suppressed GCR formation after MMS treatment. (B) GCRs were still suppressed by Ku overexpression in the lig4 strain. Mutations, which inactivate cell cycle checkpoints, abolished the suppression of GCR by Ku overexpression after MMS treatment. (C) rad24. (D) sgs1. (E) dpb11–1. (F) mec1. “+” and “−” represent the presence and absence of Ku overexpression or the treatment and no treatment of 0.05% MMS, respectively.

Growth delays or cell cycle arrests are caused by Ku overexpression in different strains. (A) Ku overexpression delays cell growth or arrests the cell cycle in wild-type or pif1-m2 strains, respectively. Pif1 overexpression either in the yku70 or the yku80 strains also arrests the cell cycle. The S phase cell cycle arrest in the pif1-m2 strain by Ku overexpression was abolished by the rad24 mutation. (B) FACS analysis demonstrates that Ku overexpression in the wild-type strain did not change the cell cycle profile. However, Ku overexpression in the pif1-m2 strain arrests the cell cycle in S phase. The rescue of cell cycle arrest in the pif1-m2 strain with Ku overexpression by the rad24 mutation was also confirmed by FACS analysis. (C) Northern blot analysis of HUG1 showed slight mRNA level increase by Ku overexpression, in contrast to the steady expression of β-actin. In contrast, there was no noticeable phosphorylation of Rad53 was observed by Ku overexpression although the phosphorylation of Rad53 was enhanced by MMS treatment. WT, wild type; Control, control plasmids, pYES3CT and pRS425, transformed. A protein name indicated in parentheses represents the protein overexpressed in the strain written in front of it. “+” and “−” represent the presence and absence of MMS treatment. The two major populations in the FACS analysis represented as black indicate G₁ and G₂/M populations. The FACS profile of pik1-m2 rad24Δ (yku70-yku80) shows one major population presented as black, and its DNA contents represent the S phase.

Ku protein functions in two different ways in dealing with DNA damage. When DNA damage (presumably DSB) occurs, Ku binds at the end of DNA and protects it, and alarms DNA damage checkpoints to protect genome. However, if DNA damage persists, Ku changes its role to recruit other DNA metabolism machinery to generate GCRs. In this stage, Ku might be modified differently compared with when it protects genome through DNA repair, telomere maintenance, and DNA damage checkpoints. Ku might interact with telomerase directly to generate de novo telomere additions or Ku could participate in the production of large deletions or translocations with ligase 4. Stars and crosses represent putative different modifications that might direct divergent roles of Ku.