No abstract

Phosphorylation of DNA-PKcs is essential for activation of DNA damage repair and for the maintenance of tissue stem cell populations.

Summary The phosphatidylinositol 3-kinase-like protein kinases (PIKK), including ATM, ATR, and DNA-PKcs, are the main kinases activated following various assaults on DNA. Although ATM and DNA-PKcs kinases are activated upon DNA double strand breaks (DSBs), evidence suggests that these kinases are rapidly phosphorylated by ATR kinase upon UV irradiation; thus these kinases may also participate in the response to replication stress. Using UV-induced replication stress, we further characterize whether ATM and DNA-PKcs kinase activities are also involved in the cellular response. Contrary to the rapid activation of the ATR-dependent pathway, ATM-dependent Chk2 and KAP-1 phosphorylations, as well as DNA-PKcs Ser2056 autophosphorylation, reach their peak level at four to eight hours after UV irradiation. The delayed kinetics of ATM and DNA-PKcs dependent phosphorylations also correlated with a surge in H2AX phosphorylation, suggesting that DSBs formation resulting from collapse of replication forks is responsible for the activation of ATM and DNA-PKcs kinases. In addition, we observed that some phosphorylation events initiated by ATR kinase in the response to UV were mediated by ATM at a later phase of the response. Furthermore, the S-phase checkpoint after UV irradiation was defective in ATM deficient cells. These results suggest that the late increase of ATM activity is needed to complement the decreasing ATR activity for maintaining a vigilant checkpoint regulation upon replication stress.

Summary The lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered the stem cell research and practice of transplantation. Major problems for allogeneic transplantation include low levels of donor engraftment and high risks of graft-versus-host disease (GVHD). Transplantation of purified allogeneic HSCs diminishes the risk of GVHD, but results in decreased engraftment. Here we show that ex vivo expanded mouse HSCs efficiently overcame the major histocompatibility complex barrier and repopulated allogeneic recipient mice. An 8-day expansion culture led to a 40-fold increase of the allograft ability of HSCs. Both increased numbers of HSCs and culture-induced elevation of expression of the immune inhibitor CD274 (B7-H1 or PD-L1) on the surface of HSCs contributed to the enhancement. Our study indicates the great potential of utilizing ex vivo expanded HSCs for allogeneic transplantation, and suggests that the immune privilege of HSCs can be modulated.

i V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs 3A ) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpinsealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs. Antigen receptor genes are assembled in developing lymphocytes through the process of V(D)J recombination (1). The V(D)J recombination reaction forms the second exon of these genes from component variable (V), joining (J), and, at some loci, diversity (D) gene segments. V(D)J recombination is initiated when the RAG-1 and RAG-2 proteins, which together form the RAG endonuclease, introduce DNA double-strand breaks (DSBs) at the border of two recombining gene segments and their associated RAG recognition sequences, termed recombination signals (RSs) (2). DNA cleavage by RAG results in two broken DNA ends with distinct structures: a blunt signal end and a coding end that is hairpin sealed by a phosphodiester bond connecting the top and bottom strands (2).