The self-renewal property of HSCs and their ability to sustain long-term hematopoiesis require that genome integrity is strictly maintained (Kenyon and Gerson, 2007
; Niedernhofer, 2008
). It is well characterized that hematopoietic cells are highly sensitive to radiation-induced DNA damage, and bone marrow aplasia is one of main causes for radiation-induced lethality. A recent study showed that HSCs, but not progenitor cells, are preferentially killed by radiation, suggesting that HSCs are among the most sensitive cell types to DNA damage (Wang et al., 2006
). HSCs in DNA repair-deficient individuals are highly susceptible to various kinds of genotoxic stresses. Genetically modified mouse models also revealed that defects in the DNA damage response result in the dysfunction of HSC maintenance. Hematopoietic failure with an early development of bone marrow hypocellularity is manifested in mice with a hypomorphic mutation of Rad50 (Rad50S/S
; Bender et al., 2002
) and mice knockout of ERCC1, a key factor in nucleotide excision repair and interstrand cross-link repair (Niedernhofer et al., 2004
; Prasher et al., 2005
). Deficiency of ATM leads to elevated levels of oxidative stress and a progressive loss of the HSC pool with age (Ito et al., 2004
). Similarly, NHEJ deficiency in Ku80 knockout mice diminishes HSC self-renewal capacity, whereas hypomorphic Lig4Y288C
mice display an age-dependent decrease in HSC numbers and bone marrow cellularity (Nijnik et al., 2007
; Rossi et al., 2007
). In contrast, HSC loss in DNA-PKcs3A/3A
mice occurs significantly earlier during embryonic development, which resembles Rad50S/S
models, though it is not clear whether there is a functional overlap between the DNA-PKcs3A
mutant protein and these two DNA repair factors in terms of HSC maintenance. On the other hand, complete DNA-PKcs deficiency leads to a decrease in HSC number (unpublished data) and repopulating ability in aged DNA-PKcs knockout mice. The distinct phenotypes in HSC maintenance between DNA-PKcs knockout and knockin mice is likely a result of their difference in DSB repair capacity, as DNA-PKcs3A/3A
cells are also defective in the HR/FA responses in addition to the diminished NHEJ activity. Thus, the combined deficiency in DSB repair renders DNA-PKcs3A/3A
cells hypersensitive to MMC and other replication stress agents. It is conceivable that the HR/FA mechanism, but not the NHEJ, is essential for replication-associated DNA damage repair and is critical for the rapid expansion of HSC populations in developing fetal liver.
DSBs are among the most dangerous types of DNA lesions and pose a great challenge to cellular survival and genome integrity maintenance. Multiple repair mechanisms, including NHEJ, HR, and additional backup pathways, have evolved to ensure that DSBs are promptly and properly eliminated from the genome. Outside V(D)J recombination events, endogenous DSBs occur primarily in the cycling cells though the conversion of single-strand lesions during replication (Vilenchik and Knudson, 2003
). From the standpoint of endogenous DSB production, we could assume that DNA-PK and its downstream NHEJ pathway, which is the predominant DSB repair mechanism in mammalian cells and is operational in all cell cycle phases, have a greater involvement in the replication-associated DNA damage response than we previously thought. However, it is not clear how DNA-PKcs and other factors of DSB repair are coordinated in response to replicative stresses, particularly in the highly proliferative cell population. Our unexpected findings from the DNA-PKcs3A
mouse model provide strong evidence that the impact of DNA-PKcs, particularly its phosphorylation at the mouse Thr2605 cluster (human Thr2609), has a role beyond the NHEJ mechanism.
It recently became clear that the competition between the NHEJ and the HR/FA pathway has important biological significance. For example, loss of the NHEJ factor 53BP1 alleviates the hypersensitivity of HR-defective Brca1 mutant cells to DNA-damaging agents and restores DSB repair by HR (Bouwman et al., 2010
; Bunting et al., 2010
). In addition, the FA pathway plays a critical role in eliminating toxic NHEJ; suppression of NHEJ alleviates the sensitivity of FA mutant cells to DNA cross-linking agents and facilitates error-free repair by HR (Adamo et al., 2010
; Pace et al., 2010
). Similarly, our data suggest that the DNA-PKcs3A
mutant protein interferes with the HR pathway, which could explain the sensitivity to replication damage and the severity of hematopoietic failure.
We have previously reported that the human Thr2609 cluster is phosphorylated directly by the ATR kinase upon replication stress (Yajima et al., 2006
). This ATR-dependent DNA-PKcs phosphorylation virtually facilitates processing of replication-associated DNA lesions through the HR/FA machinery. It is likely that, in such a repair process, the shift from error-prone end joining to faithful repair by the HR/FA is partially achieved by ATR-dependent DNA-PKcs phosphorylation. In support of this, a similar stem cell defect was observed in conditional ATR knockout mice (Ruzankina et al., 2007
). It is not clear yet how DNA-PKcs phosphorylation at the Thr2609 cluster promotes HR/FA activities. One possible explanation is that Thr2609 cluster phosphorylation changes the conformation of DSB-bound DNA-PKcs and releases DSB ends to the HR/FA machineries (Meek et al., 2008
The loss of HSCs in DNA-PKcs3A/3A mice is primarily caused by p53-mediated apoptotic response, as the p53-null mutation reinstates HSC reserves in DNA-PKcs3A/3A mice. In addition to HSC loss, elevation of genotoxic stress and p53 activation was found in other proliferating tissues as evident from intestinal crypt cell apoptosis and epidermal hyperpigmentation. These results reiterate that DNA-PKcs phosphorylation at the human Thr2609/mouse Thr2605 cluster is critical for replication stress response and the maintenance of tissue homeostasis. The tissue-specific outcome of genotoxic stress imposed by the DNA-PKcs3A mutant protein or DNA-PKcs dysfunction may depend on cell type–specific apoptosis/senescence and the compensation capacity from varied sizes of stem cell pools in different tissues and organs.
Gene mutations in the FA protein complex and HR components have been indentified in human FA syndrome characterized by congenital bone marrow failure, short stature, abnormal skin pigmentation, reduced fertility, hypersensitivity toward DNA cross-link damages, and increased genomic instability (Moldovan and D’Andrea, 2009
). The mechanism underlying the clinical findings of FA remains unclear, given the fact that none of the current mouse models for HR and FA systematically recapitulated these pathophysiological changes. It is interesting to note that the DNA-PKcs3A/3A
mouse resembles human FA syndromes more than FA mouse models. The hallmarks of FA, including aplastic anemia, abnormal skin pigmentation, MMC sensitivity, and radial chromosome formation with MMC treatment, are all observed in DNA-PKcs3A/3A
mice, suggesting that, in human patients, FA mutations also likely affect the stem cell compartment during embryo development and activate p53, which generates pathological changes in multiple organs. Indeed, p53 has also been shown to be up-regulated in FA cells, and it has been speculated that the p53-dependent apoptotic pathway may contribute to the loss of stem cells in bone marrow (Kennedy and D’Andrea, 2005
; Rani et al., 2008
). It is interesting to note that the human dyskeratosis congenita syndrome, which is caused by telomerase mutations, also displays bone marrow failure and hyper skin pigmentation characters. Conversely, mice with combined mutations on telomerase and Pot1b, a component of the telomere-protecting shelterin complex, exhibit dyskeratosis congenita–like characteristics, including bone marrow failure and footpad hyperpigmentation (Hockemeyer et al., 2008
; He et al., 2009
). This similarity to DNA-PKcs3A/3A
mice suggests a common stress response among different congenital bone marrow failure diseases and warrants further investigation of DNA-PKcs phosphorylation on telomere maintenance.