The phosphatidyl inositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and ATM-and Rad3-related (ATR) regulate parallel damage response signalling pathways. ATM is reported to be activated by DNA double-strand breaks (DSBs), whereas ATR is recruited to single-stranded regions of DNA. Although the two pathways were considered to function independently, recent studies have demonstrated that ATM functions upstream of ATR following exposure to ionising radiation (IR) in S/G2. Here, we show that ATM phosphorylation at Ser1981, a characterised autophosphorylation site, is ATR-dependent and ATM-independent following replication fork stalling or UV treatment. In contrast to IR-induced ATM-S1981 phosphorylation, UV-induced ATM-S1981 phosphorylation does not require the Nbs1 C-terminus or Mre11. ATRdependent phosphorylation of ATM activates ATM phosphorylation of Chk2, which has an overlapping function with Chk1 in regulating G2/M checkpoint arrest. Our findings provide insight into the interplay between the PIKK damage response pathways.

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiationand hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR-and ATMdependent signalling. We propose that the NBS clinical features represent the result of these combined defects.

DNA ligase IV functions in DNA non-homologous endjoining, in V(D)J recombination, and during brain development. We previously reported a homozygous mutation (R278H) in DNA ligase IV in a developmentally normal leukemia patient who overresponded to radiotherapy. The impact of this hypomorphic mutation has been evaluated using cellular, biochemical, and structural approaches. Structural modeling using T7 DNA ligase predicts that the activity and conformational stability of the protein is likely to be impaired. We show that wild type DNA ligase IV-Xrcc4 is an efficient double-stranded ligase with distinct optimal requirements for adenylate complex formation versus rejoining. The mutation impairs the formation of an adenylate complex as well as reducing the rejoining activity. Additionally, it imparts temperature-sensitive activity to the protein consistent with the predictions of the structural modeling. At the cellular level, the mutation confers a unique V(D)J recombination phenotype affecting the fidelity of signal joint formation with little effect on the frequency of the reaction. These findings suggest that hypomorphic mutations in ligase IV may allow normal development but confer marked radiosensitivity.

XLF-Cernunnos (XLF) is a component of the DNA ligase IV–XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.

The DNA ligase IV⅐XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity. DNA non-homologous end-joining (NHEJ)1 is the major mechanism for the repair of DNA double-strand breaks in mammalian cells and rejoins breaks induced during V(D)J recombination. Five proteins function directly in the end-joining process in mammalian cells (1, 2), namely DNA ligase IV and XRCC4, and the DNA-dependent protein kinase complex (DNA-PK), which comprises two subunits of the Ku heterodimer and a large catalytic subunit, DNA-PKcs. Recently, Artemis has also been shown to play some role in the DNA NHEJ process (3, 4).DNA ligase IV has a conserved ligase domain at its N terminus and two BRCT domains at its C terminus. DNA ligase IV forms a tight complex with XRCC4 (LX) via an interaction that requires the region between the two BRCT domains (5). Recently, mutations in DNA ligase IV were found in patients displaying radiosensitivity, immunodeficiency, and developmental delay (6). Although LX plays a unique role in NHEJ, it has been reported to be an inefficient double-stranded (ds) ligase and specifically is less effective than either of the two more abundant mammalian ligases, DNA ligases I and II (7-10). However, using similar molar ratios of enzyme and substrate to those used in studies reporting inefficient ds ligation by LX, we found that baculovirus-expressed LX is a highly efficient ds ligase (7-11). Here, we examine the basis underlying these contradictory findings and show that LX has specific substrate length requirements. Using longer substrates than those used previously, we show that the LX comple...