The Ataxia Telangiectasia-Mutated (ATM) protein kinase is recruited to sites of double-strand DNA breaks by the Mre11/Rad50/Nbs1 (MRN) complex, which also facilitates ATM monomerization and activation. MRN exists in at least two distinct conformational states, dependent on ATP binding and hydrolysis by the Rad50 protein. Here we use an ATP analog-sensitive form of ATM to determine that ATP binding, but not hydrolysis, by Rad50 is essential for MRN stimulation of ATM. Mre11 nuclease activity is dispensable, although some mutations in the Mre11 catalytic domain block ATM activation independent of nuclease function, as does the mirin compound. The coiled-coil domains of Rad50 are important for the DNA binding ability of MRN and are essential for ATM activation, but loss of the zinc hook connection can be substituted by higher levels of the complex. Nbs1 binds to the "closed" form of the MR complex, promoted by the zinc hook and by ATP binding. Thus the primary role of the hook is to tether Rad50 monomers together, promoting the association of the Rad50 catalytic domains into a form that binds ATP and also binds Nbs1. Collectively, these results show that the ATP-bound form of MRN is the critical conformation for ATM activation.
The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.
The prostate tumor suppressor NKX3.1 augments response to DNA damage and enhances survival after DNA damage. Within minutes of DNA damage, NKX3.1 undergoes phosphorylation at tyrosine 222, which is required for a functional interaction with ataxia telangiectasia mutated (ATM) kinase. NKX3.1 binds to the N-terminal region of ATM, accelerates ATM activation, and hastens the formation of γhistone2AX. NKX3.1 enhances DNA-dependent ATM kinase activation by both the MRN complex and H2O2 in a DNA-damage-independent manner. ATM, bound to the NKX3.1 homeodomain, phosphorylates NKX3.1, leading to ubiquitination and degradation. Thus, NKX3.1 and ATM have a functional interaction leading to ATM activation and then NKX3.1 degradation in a tightly regulated DNA damage response specific to prostate epithelial cells. These findings demonstrate a mechanism for the tumor-suppressor properties of NKX3.1, demonstrate how NKX3.1 may enhance DNA integrity in prostate stem cells and may help to explain how cells differ in their sensitivity to DNA damage.
Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.
The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double-strand break signalling in human cells. Here, we compare the DNA-binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single-strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single-molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates. Nevertheless, the SOSS1 complex is uniquely capable of promoting interaction of human Exo1 with double-strand DNA ends and stimulates its activity independently of the MRN complex in vitro. Both MRN and SOSS1 also act to mitigate the inhibitory action of the Ku70/80 heterodimer on Exo1 activity in vitro. These results may explain why SOSS complexes do not localize with RPA to replication sites in human cells, yet have a strong effect on double-strand break resection and homologous recombination.
The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.
