Significance of DNA-PKcs Phosphorylation

When double-stranded breaks occur, the fast DSB-binding of the Ku70/Ku80 homodimer recruits and promotes the kinase activity of DNA-PKcs and its phosphorylation at the Thr2609 cluster and Ser2056. DNA-PKcs phosphorylation at the Thr2609 cluster region is particularly critical for DSB repair and cellular resistance to ionizing radiation. Although the Thr2609 cluster was initially identified in vitro upon DNA-PKcs activation, further examination revealed that Thr2609 cluster phosphorylation is primarily regulated by the ataxia-telangiectasia-mutated (ATM) and the Rad3-related (ATR) kinases in response to DNA damage. On the contrary, DNA-PKcs phosphorylation at Ser2056 is absolutely mediated by DNA-PKcs autophosphorylation, making it an excellent marker to monitor DNA-PKcs activation in vivo.

Kaplan-Meier survival curves for untreated and bone-marrow-transplanted DNA-PKcs3A/3A mice

The critical function of DNA-PKcs phosphorylation at the Thr2609 cluster has led us to develop a knock-in mouse model harboring three alanine substitutions (3A mutation in brief) at the mouse equivalent Thr2605 cluster. In sharp contrast to DNA-PKcs knockout (DNA-PKcs-/-) and DNA-PKcs-deficient SCID mice, homozygous DNA-PKcs3A/3A mice died prematurely within 1-2 months after birth owing to congenital bone marrow failure and loss of hematopoietic stem cells (HSCs). In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and critical function of DNA-PKcs for the maintenance of diverse populations of tissue stem cells in mice.

Stained cells
Bone marrow hypo-cellularity, increased intestinal crypt apoptosis, and skin hyperpigmentation in DNA-PKcs3A/3A mice (left panel). Overlapping of γH2AX foci with leading strand telomeres in chromosome orientation fluorescence in situ hybridization (right panel).

The premature death of DNA-PKcs3A/3A mice could be rescued by bone marrow transplantation (BMT). Surprisingly, an increased incidence of spontaneous tumors was observed in BMT-rescued DNA-PKcs3A/3A mice. On the contrary, solid tumor development was rarely observed in DNA-PKcs-/- mice. Upon further investigation, we observed significantly elevated frequencies of telomere fusion events in DNA-PKcs3A/3A cells and spontaneous γH2AX foci overlapped with telomeres during mitosis, indicating impairment of telomere replication and maturation.

Our analyses indicate that the functional DNA-PKcs Thr2609 cluster is required for telomere maintenance to prevent genome instability and cancer development.