Rajashree A. Deshpande

ATM activation in the presence of oxidative stress

The Ataxia-Telangiectasia mutated (ATM) kinase is regarded as the major regulator of the cellular response to DNA double strand breaks (DSBs). In response to DSBs, ATM dimers dissociate into active monomers in a process promoted by the Mre11-Rad50-Nbs1 (MRN) complex. ATM can also be activated by oxidative stress directly in the form of exposure to H2O2. The active ATM in this case is a disulfide-crosslinked dimer containing 2 or more disulfide bonds. Mutation of a critical cysteine residue in the FATC domain involved in disulfide bond formation specifically blocks ATM activation by oxidative stress. Here we show that ATM activation by DSBs is inhibited in the presence of H2O2 because oxidationblocks the ability of MRN to bind to DNA. However, ATM activation via direct oxidation by H2O2 complements the loss of MRN/DSB-dependent activation and contributes significantly to the overall level of ATM activity in the presence of both DSBs and oxidative stress.

Ataxia Telangiectasia-Mutated (ATM) Kinase Activity Is Regulated by ATP-driven Conformational Changes in the Mre11/Rad50/Nbs1 (MRN) Complex

Background: The Mre11/Rad50/Nbs1 (MRN) complex regulates DNA repair and signaling through the Ataxia Telangiectasia-Mutated (ATM) kinase. Results: ATM activation requires ATP binding by Rad50 and the coiled-coils but not ATP hydrolysis, zinc hook connection, or Mre11 nuclease function. Conclusion: The ATP-bound form of MRN with Rad50 catalytic domains engaged is the form that activates ATM. Significance: ATP-driven changes in MRN conformation control ATM signaling. 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.

EXD2 promotes homologous recombination by facilitating DNA end resection

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN–CtIP-dependent end resection, which generates the 3′ single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3′–5′ exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.

The Mre11/Rad50/Nbs1 complex: Recent insights into catalytic activities and ATP-driven conformational changes

DNA double-strand breaks (DSBs) can arise from internal or external sources of damage, and the rapid detection, processing, and repair of this damage is important for cell viability. Failure to repair DNA damage can result in genomic instability, ultimately increasing the frequency of lymphoid disorders, neurodegeneration, and cancer. The Mre11-Rad50-Nbs1 (Xrs2) complex plays a central and critical role in detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11/Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes (Lamarche et al., 2010; Stracker and Petrini, 2011). The importance of this complex is emphasized by the fact that deletion of any of the three components results in embryonic lethality in mice and loss of proliferative activity in embryonic stem cells (Buis et al., 2008; Luo et al., 1999; Xiao and Weaver, 1997; Zhu et al., 2001) which is likely related to the role of MRN/X in homologous recombination. Repair of DSBs by homologous recombination involves replication of the broken region using an undamaged template, usually a sister chromatid. Deletions of other genes important for homologous forms of repair also exhibit early embryonic lethality, including Rad51, BRCA1, BRCA2, and CtBP-interacting protein (CtIP)(Chen et al., 2005b; Gowen et al., 1996; Lim and Hasty, 1996; Sharan et al., 1997). Hypomorphic mutations in MRN components result into developmental and neurodegenerative disorders in humans, including Ataxia-Telangiectasia-Like Disorder (ATLD), Nijmegen Breakage Syndrome (NBS), and NBS-like syndrome (Matsumoto et al., 2011; Stewart et al., 1999; Varon et al., 1998; Waltes et al., 2009), which are related, at least in part, to the role of MRN/X in the activation of cell-cycle checkpoints through the Ataxia-Telangiectasia-Mutated (ATM) protein kinase (Lee and Paull, 2007; Shiloh and Ziv, 2013). The roles of MRN/X also extend to the processing of DSBs during meiosis, for which it is essential, and to telomere maintenance (Borde, 2007; Lamarche et al., 2010). Repair of DSBs is achieved through two broadly-defined groups of pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR) (Krogh and Symington, 2004). The choice between these pathways primarily depends on the cell-cycle phase and the complexity of the damage generated at the break site (Chapman et al., 2012; Schipler and Iliakis, 2013). In the classical NHEJ pathway, ends are bound by the Ku70–Ku80 heterodimer/DNA-dependent protein kinase catalytic subunit (DNA-PKcs) complex which recruits additional factors involved in end modifications and gap filling. DNA ends are ultimately ligated by the NHEJ-specific DNA ligase IV complex (Deriano and Roth, 2013). In mammalian cells, the C-NHEJ pathway is not dependent on the MRN complex, although in budding yeast MRX contributes to NHEJ pathway through interactions with Ku70-Ku80 and DNA Lig4 complexes (Lewis and Resnick, 2000). The MRN complex, in conjunction with CtIP/Sae2, also regulates the alternative NHEJ (A-NHEJ or MMEJ), which utilizes short microhomologies and can result in large deletions (Lee and Lee, 2007; Yun and Hiom, 2009). In mammalian cells MRN was also shown to interact with DNA ligaseIIIα/Xrcc1, the ligase complex implicated in alternative NHEJ, stimulating intermolecular ligation (Della-Maria et al., 2011). In contrast to NHEJ, HR requires the 5′–3′ resection of dsDNA to generate single-stranded DNA tails, a process that is initiated by the MRN complex and CtIP (You and Bailis, 2010). Extensive resection is perfomed by exonuclease 1 (Exo1), and Dna2 (Symington and Gautier, 2011), whose activities are also promoted by MRN (Cejka et al., 2010; Nicolette et al., 2010; Niu et al., 2010; Yang et al., 2013; Zhou et al., 2014; Zhou and Paull, 2013). 3′ ssDNA tails thus generated are bound by replication protein A (RPA), which activates ATM- and Rad3-Related (ATR), promoting replication checkpoint arrest and stabilization of replication forks (Zeman and Cimprich, 2014). RPA on these 3′ ssDNA tails is then exchanged for Rad51 to create Rad51 filaments that catalyze homology search and strand invasion, ultimately priming DNA synthesis and resolution of repair intermediates. The MRN complex plays important and diverse roles in DNA double-strand break repair and signaling. Here we review recent evidence elucidating the structures and regulation of the Mre11/Rad50 complex, focusing primarily on the enzymatic activities of MRN and the role of ATP-driven conformational changes in Rad50.

Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites

Abstract The Mre11–Rad50–Nbs1(Xrs2) (MRN/X) complex is critical for the repair and signaling of DNA double strand breaks. The catalytic core of MRN/X comprised of the Mre11 nuclease and Rad50 adenosine triphosphatase (ATPase) active sites dimerizes through association between the Rad50 ATPase catalytic domains and undergoes extensive conformational changes upon ATP binding. This ATP-bound ‘closed’ state promotes binding to DNA, tethering DNA ends and ATM activation, but prevents nucleolytic processing of DNA ends, while ATP hydrolysis is essential for Mre11 endonuclease activity at blocked DNA ends. Here we investigate the regulation of ATP hydrolysis as well as the interdependence of the two functional active sites. We find that double-stranded DNA stimulates ATP hydrolysis by hMRN over ∼20-fold in an end-dependent manner. Using catalytic site mutants to create Rad50 dimers with only one functional ATPase site, we find that both ATPase sites are required for the stimulation by DNA. MRN-mediated endonucleolytic cleavage of DNA at sites of protein adducts requires ATP hydrolysis at both sites, as does the stimulation of ATM kinase activity. These observations suggest that symmetrical engagement of the Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells.

DNA-PKcs promotes DNA end processing

DNA-dependent Protein Kinase (DNA-PK) coordinates the repair of double-strand breaks through non-homologous end joining, the dominant repair pathway in mammalian cells. Breaks can also be resolved through homologous recombination during S/G2 cell cycle phases, initiated by the Mre11-Rad50-Nbs1 (MRN) complex and CtIP-mediated resection of 59 strands. The functions of DNA-PK are considered to be end-joining specific, but here we demonstrate that human DNA-PK also plays an important role in the processing of DNA double-strand breaks. Using ensemble biochemistry and single-molecule approaches, we show that the MRN complex in cooperation with CtIP is stimulated by DNA-PK to perform efficient endonucleolytic processing of DNA ends in physiological conditions. This activity requires both CDK and ATR phosphorylation of CtIP. These unexpected results could explain the absence of DNA-PK deletion mutations in the human population, as homologous recombination is an essential process in mammals.The repair of DNA double-strand breaks occurs through non-homologous end joining or homologous recombination in vertebrate cells - a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from non-homologous end joining to homologous recombination by facilitating DNA-PK removal. One Sentence Summary DNA-dependent protein kinase, an enzyme critical for non-homologous repair of DNA double-strand breaks, also stimulates end processing for homologous recombination.

Erratum: ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling (EMBO Journal (2014) 33 (482-500)) DOI 10.1002/embj.201386100

Thank you for submitting your manuscript on ATP-driven Rad50 conformational changes for consideration by The EMBO Journal. We have now received comments from three expert referees who evaluated the study. I am pleased to inform that all of them consider this work of interest, and for the most part well-conducted and well-presented, and we should therefore be happy to consider a revised version of this manuscript further for publication in our journal. Nevertheless, all referees do raise a number of specific issues that would need to be satisfactorily addressed prior to acceptance. As you will see from the reports, while this amounts to a considerable number of points, most of them appear to be well taken and straightforward, and referring to specific issues rather than major conceptual concerns. I would therefore like to invite you to revise the study taking into account the various referee comments and suggestions, and to resubmit a revised manuscript together with a detailed point-by-point response letter. In doing so, please also make sure to expand the discussion (as requested by referee 3) to better discuss your results in light of relevant previously published findings, including those in your own recent paper (Lee et al, JBC March 2013).