Spontaneous tumor development in bone marrow-rescued DNA-PKcs(3A/3A) mice due to dysfunction of telomere leading strand deprotection.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at the Thr2609 cluster is essential for its complete function in DNA repair and tissue stem cell homeostasis. This phenomenon is demonstrated by congenital bone marrow failure occurring in DNA-PKcs(3A/3A) mutant mice, which require bone marrow transplantation (BMT) to prevent early mortality. Surprisingly, an increased incidence of spontaneous tumors, especially skin cancer, was observed in adult BMT-rescued DNA-PKcs(3A/3A) mice. Upon further investigation, we found that spontaneous γH2AX foci occurred in DNA-PKcs(3A/3A) skin biopsies and primary keratinocytes and that these foci overlapped with telomeres during mitosis, indicating impairment of telomere replication and maturation. Consistently, we observed significantly elevated frequencies of telomere fusion events in DNA-PKcs(3A/3A) cells as compared with wild-type and DNA-PKcs-knockout cells. In addition, a previously identified DNA-PKcs Thr2609Pro mutation, found in breast cancer, also induces a similar impairment of telomere leading-end maturation. Taken together, our current analyses indicate that the functional DNA-PKcs T2609 cluster is required to facilitate telomere leading strand maturation and prevention of genomic instability and cancer development.

DNA-PKcs activates the Chk2-Brca1 pathway during mitosis to ensure chromosomal stability.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is known to have a critical role in DNA double-strand break repair. We have previously reported that DNA-PKcs is activated when cells enter mitosis and functions in mitotic spindle assembly and chromosome segregation. Here we report that DNA-PKcs is the upstream regulator of the Chk2-Brca1 pathway, which impacts microtubule dynamics, kinetochore attachment and chromosomal segregation in mitosis. Downstream from Chk2, Brca1 promotes monoubiquitination of γ-tubulin to inhibit microtubule nucleation and growth. We found that DNA-PKcs is essential for mitotic Chk2 phosphorylation at Thr68. As in Chk2- and Brca1-deficient cells, loss of DNA-PKcs resulted in chromosome misalignment and lagging during anaphase owing to elevation in microtubule dynamics. Importantly, these mitotic aberrations in DNA-PKcs-defective cells were alleviated by the overexpression of phosphomimetic Chk2 or Brca1 mutant proteins but not their wild-type counterparts. Taken together, these results demonstrate that DNA-PKcs regulates mitotic spindle organization and chromosomal instability via the Chk2-Brca1 signaling pathway.