Investigating the role of the KU-binding motif in DNA repair

The KU heterodimer (KU70/80) regulates the processing and repair of DNA double-strand breaks (DSBs). Previous work has revealed that the KU80 vWA-like domain interacts with a short peptide motif that is present in a range of DNA repair proteins and is denoted the KU- binding motif (KBM). Here, I employed mass spectrometry and cell biology to identify and characterise novel KBM-containing proteins, and to define the importance of KBM-binding for KU80 function.

To determine the importance of the KU80 vWA-like domain, I developed stable human U2OS cells expressing transgenes encoding wild-type or mutant KU80 that cannot bind KBM- containing proteins. Endogenous KU80 was successfully deleted in the U2OS cells expressing a wild-type KU80 transgene, but not in U2OS cells expressing the mutated transgene, suggesting that KBM-binding is essential for survival in U2OS cells. Therefore, I employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells, which unlike human cells, are viable. NHEJ efficiency was normal in Xrs-6 cells expressing mutant KU80, following ionising radiation, but these cells accumulated pan-nuclear yH2AX in S-/G2-phase and exhibited hypersensitivity to DNA replication stress induced by camptothecin and hydroxyurea. These data suggest that KBM- binding to KU80 is required for the processing and/or repair of single-ended DSBs.

To identify the KBM-containing protein/s responsible for these phenotypes, I measured phosphorylation of RPA Ser4/8, which is catalysed by DNA-PKcs, and observed that this was reduced in Xrs-6 cells expressing KU80 that harbours a mutated KBM-binding domain. I also identified, for the first time, a KBM in DNA-PKcs. Furthermore, depletion of WRN, a helicase/exonuclease that harbours two KBM motifs, recapitulated the camptothecin induced yH2AX phenotype.

In summary, my data identifies a role for the KBM-binding domain of KU80 in the response and resistance of Chinese Hamster Ovary cells to DNA replication stress, and implicate KBMs in DNA-PKcs and WRN as effectors of this role.