DNA damage during meiosis induces chromatin remodeling and synaptonemal complex disassembly

DNA damage to the germline genome must be accurately repaired to ensure transmission of intact genetic information to following generations. Meiosis presents challenges to the DNA damage response (DDR) because it universally requires changes to chromosome structure that can affect DNA repair outcomes. We report the existence of a meiotic DDR at chromosome axes that results in chromatin remodeling, synaptonemal complex disassembly, and axis separation in response to irradiation at late pachytene stages in C. elegans. The axis component HTP-3 is required for germline acquisition of H2AacK5, an axis-specific chromatin mark that is DNA damage responsive. Irradiated wild-types show reduction of H2AacK5 and axis separation that are dependent on the acetyltransferase MYS-1/TIP60. Restoration of H2AacK5 levels requires ATM-1 kinase and correlates with resynapsis. We propose that the meiotic DDR involves early chromatin remodeling at chromosome axes to dismantle structures promoting interhomolog recombination and facilitate efficient nonhomolog-based repair before pachytene exit.     

Differential regulation of MRN (Mre11-Rad50-Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy.     

DNA replication-dependent nuclear dynamics of the Mre11 complex

The Mre11 complex undergoes dramatic relocalization in the nuclei of gamma-irradiated and replicating human cells. In this study, we examined Mre11 complex localization and chromatin association in synchronous cultures to examine the molecular determinants of relocalization. The data indicate that the complex is deposited on chromatin in an S phase-specific manner. Mre11 complex chromatin association in S phase was resistant to detergent extraction, in contrast to that in gamma-irradiated cells. The complex exhibits extensive colocalization with proliferating cell nuclear antigen throughout S phase, and chromatin loading is enhanced by replication fork stalling, suggesting that the replication fork is a site of Mre11 complex chromatin loading. This is supported by the observation that the complex localized to single-stranded DNA arising in hydroxyurea-treated cells. Although the Mre11 complex appears to function as a DNA damage sensor, limited colocalization with Brca1 or gamma-H2AX was observed, arguing that neither DNA damage nor gamma-H2AX is required for Mre11 complex chromatin loading. These data provide a potential molecular basis for promotion of sister chromatid association and recombination by the Mre11 complex as well as for ATM-Mre11 complex-dependent activation of cell cycle checkpoints.     

DNA damage-dependent nuclear dynamics of the Mre11 complex

The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.     

Mre11-Rad50 complex crystals suggest molecular calisthenics

Recently published crystal structures of different Mre11 and Rad50 complexes show the arrangement of these proteins and imply dramatic ligand-induced rearrangements with important functional consequences.     

Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response

BACKGROUND: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. RESULTS: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. CONCLUSIONS: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.     

Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex

Results reported here indicate that adenovirus 5 exploits the cellular aggresome response to accelerate inactivation of MRE11-RAD50-NBS1 (MRN) complexes that otherwise inhibit viral DNA replication and packaging. Aggresomes are cytoplasmic inclusion bodies, observed in many degenerative diseases, that are formed from aggregated proteins by dynein-dependent retrograde transport on microtubules to the microtubule organizing center. Viral E1B-55K protein forms aggresomes that sequester p53 and MRN in transformed cells and in cells transfected with an E1B-55K expression vector. During adenovirus infection, the viral protein E4orf3 associates with MRN in promyelocytic leukemia protein nuclear bodies before MRN is bound by E1B-55K. Either E4orf3 or E4orf6 is required in addition to E1B-55K for E1B-55K aggresome formation and MRE11 export to aggresomes in adenovirus-infected cells. Aggresome formation contributes to the protection of viral DNA from MRN activity by sequestering MRN in the cytoplasm and greatly accelerating its degradation by proteosomes following its ubiquitination by the E1B-55K/E4orf6/elongin BC/Cullin5/Rbx1 ubiquitin ligase. Our results show that aggresomes significantly accelerate protein degradation by the ubiquitin-proteosome system. The observation that a normal cellular protein is inactivated when sequestered into an aggresome through association with an aggresome-inducing protein has implications for the potential cytotoxicity of aggresome-like inclusion bodies in degenerative diseases.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy

The RAD50 gene of Saccharomyces cerevisiae is one of several genes required for recombinational repair of double-strand DNA breaks during vegetative growth and for initiation of meiotic recombination. Rad50 forms a complex with two other proteins, Mre11 and Xrs2, and this complex is involved in double-strand break formation and processing. Rad50 has limited sequence homology to the structural maintenance of chromosomes (SMC) family of proteins and shares the same domain structure as SMCs: N- and C-terminal globular domains separated by two long coiled-coils. However, a notable difference is the much smaller non-coil hinge region between the two coiled-coils. We report here a structural analysis of full-length S. cerevisiae Rad50, alone and in a complex with yeast Mre11 by electron microscopy. Our results confirm that yeast Rad50 does have the same antiparallel coiled-coil structure as SMC proteins, but with no detectable globular hinge domain. However, the molecule is still able to bend sharply in the middle to bring the two catalytic domains together, indicating that the small hinge domain is flexible. We also demonstrate that Mre11 binds as a dimer between the catalytic domains of Rad50, bringing the nuclease activities of Mre11 in close proximity to the ATPase and DNA binding activities of Rad50.     

DNA lesion-specific co-localization of the Mre11/Rad50/Nbs1 (MRN) complex and replication protein A (RPA) to repair foci

The DNA damage response, triggered by DNA replication stress or DNA damage, involves the activation of DNA repair and cell cycle regulatory proteins including the MRN (Mre11, Rad50, and Nbs1) complex and replication protein A (RPA). The induction of replication stress by hydroxyurea (HU) or DNA damage by camptothecin (CAMPT), etoposide (ETOP), or mitomycin C (MMC) led to the formation of nuclear foci containing phosphorylated Nbs1. HU and CAMPT treatment also led to the formation of RPA foci that co-localized with phospho-Nbs1 foci. After ETOP treatment, phospho-Nbs1 and RPA foci were detected but not within the same cell. MMC treatment resulted in phospho-Nbs1 foci formation in the absence of RPA foci. Consistent with the presence or absence of RPA foci, RPA hyperphosphorylation was present following HU, CAMPT, and ETOP treatment but absent following MMC treatment. The lack of co-localization of phospho-Nbs1 and RPA foci may be due to relatively shorter stretches of single-stranded DNA generated following ETOP and MMC treatment. These data suggest that, even though the MRN complex and RPA can interact, their interaction may be limited to responses to specific types of lesions, particularly those that have longer stretches of single-stranded DNA. In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions.     

Functional interactions between Sae2 and the Mre11 complex

The Mre11 complex functions in double-strand break (DSB) repair, meiotic recombination, and DNA damage checkpoint pathways. Sae2 deficiency has opposing effects on the Mre11 complex. On one hand, it appears to impair Mre11 nuclease function in DNA repair and meiotic DSB processing, and on the other, Sae2 deficiency activates Mre11-complex-dependent DNA-damage-signaling via the Tel1-Mre11 complex (TM) pathway. We demonstrate that SAE2 overexpression blocks the TM pathway, suggesting that Sae2 antagonizes Mre11-complex checkpoint functions. To understand how Sae2 regulates the Mre11 complex, we screened for sae2 alleles that behaved as the null with respect to Mre11-complex checkpoint functions, but left nuclease function intact. Phenotypic characterization of these sae2 alleles suggests that Sae2 functions as a multimer and influences the substrate specificity of the Mre11 nuclease. We show that Sae2 oligomerizes independently of DNA damage and that oligomerization is required for its regulatory influence on the Mre11 nuclease and checkpoint functions.     

Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.     

Oxidative stress induces cell cycle-dependent Mre11 recruitment,ATM and Chk2 activation and histone H2AX phosphorylation

DNA damage response recruits complex molecular machinery involved in DNA repair, arrest of cell cycle progression, and potentially in activation of apoptotic pathway. Among the first responders is the Mre11- (MRN) complex of proteins (Mre11, Rad50, Nbs1), essential for activation of ATM; the latter activates checkpoint kinase 2 (Chk2) and phosphorylates histone H2AX. In the present study the recruitment of Mre11 and phosphorylation of ATM, Chk2 and H2AX (γH2AX) detected immunocytochemically were measured by laser scanning cytometry to assess kinetics of these events in A549 cells treated with H2O2. Recruitment of Mre11 was rapid, peaked at 10 min of exposure to the oxidant, and was of similar extent in all phases of the cell cycle. ATM and Chk2 activation as well as H2AX phosphorylation reached maximum levels after 30 min of treatment with H2O2; the extent of phosphorylation of each was most prominent in S-, less in G₁-, and the least in G₂M- phase cells. A strong correlation between activation of ATM and Chk2, measured in the same cells, was seen in all phases of the cycle. In untreated cells activated Chk2 and Mre11 were distinctly present in centrosomes while in interphase cells they had characteristic punctate nuclear localization. The punctate expression of activated Chk2 both in untreated and H₂O₂ treated cells was accentuated when measured as maximal pixel rather than integrated value of immunofluorescence (IF) per nucleus, and was most pronounced in G₁ cells, likely reflecting the function of Chk2 in activating Cdc25A. Subpopulations of G₁ and G₂M cells with strong maximal pixel of Chk2-Thr68P IF in association with centrosomes were present in untreated cultures. Cytometric multiparameter assessment of the DNA damage response utilizing quantitative image analysis that allows one to measure inhomogeneity of fluorochrome distribution (e.g. maximal pixel) offers unique advantage in studies of the response of different cell constituents in relation to cell cycle position.     

Biochemical characterization of bacteriophage T4 Mre11-Rad50 complex

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.     

Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure

We reported that stoichiometric phosphorylation by either cAMP-dependent protein kinase or protein kinase C induces disassembly of vimentin filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato, C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263, 5970-5978]. In the present work, we attempted to identify the sites of vimentin phosphorylated by each protein kinase. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were specifically phosphorylated by protein kinase C, whereas Ser-46 was phosphorylated preferentially by cAMP-dependent protein kinase. Both kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific phosphorylation sites for protein kinase C are mostly located close to the amino-terminal side of arginine while those for cAMP-dependent protein kinase are located close to the carboxyl-terminal side of arginine. The phosphorylation sites exclusively occur in the amino-terminal non-alpha-helical head domain, particularly at the beta-turn region. These results provide clues to the molecular mechanisms of phosphorylation-dependent disassembly of vimentin filaments.     

Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly

Cells utilize dynamic interactions with the extracellular matrix to adapt to changing environmental conditions. Thrombospondin 1 (TSP1) induces focal adhesion disassembly and cell migration through a sequence (hep I) in its heparin-binding domain signaling through the calreticulin-low density lipoprotein receptor-related protein receptor complex. This involves the Galphai-dependent activation of ERK and phosphoinositide (PI) 3-kinase, both of which are required for focal adhesion disassembly. Focal adhesion kinase (FAK) regulates adhesion dynamics, acting in part by modulating RhoA activity, and FAK is implicated in ERK and PI 3-kinase activation. In this work, we sought to determine the role of FAK in TSP1-induced focal adhesion disassembly. TSP1/hep I does not stimulate focal adhesion disassembly in FAK knockout fibroblasts, whereas re-expressing FAK rescues responsiveness. Inhibiting FAK signaling through FRNK or FAK Y397F expression in endothelial cells also abrogates this response. TSP1/hep I stimulates a transient increase in FAK phosphorylation that requires calreticulin and Galphai, but not ERK or PI 3-kinase. Hep I does not activate ERK or PI 3-kinase in FAK knockout fibroblasts, suggesting activation occurs downstream of FAK. TSP1/hep I stimulates RhoA inactivation with kinetics corresponding to focal adhesion disassembly in a FAK, ERK, and PI 3-kinase-dependent manner. Furthermore, hep I does not stimulate focal adhesion disassembly in cells expressing constitutively active RhoA, suggesting that RhoA inactivation is required for this response. This is the first work to illustrate a connection between FAK phosphorylation in response to a soluble factor and RhoA inactivation, as well as the first report of PI 3-kinase and ERK in FAK regulation of RhoA activity.     

Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex

DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. We propose that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome.     

DNA damage induces nucleoid compaction via the Mre11‐Rad50 complex in the archaeon Haloferax volcanii

In prokaryotes the genome is organized in a dynamic structure called the nucleoid, which is embedded in the cytoplasm. We show here that in the archaeon Haloferax volcanii, compaction and reorganization of the nucleoid is induced by stresses that damage the genome or interfere with its replication. The fraction of cells exhibiting nucleoid compaction was proportional to the dose of the DNA damaging agent, and results obtained in cells defective for nucleotide excision repair suggest that breakage of DNA strands triggers reorganization of the nucleoid. We observed that compaction depends on the Mre11‐Rad50 complex, suggesting a link to DNA double‐strand break repair. However, compaction was observed in a radA mutant, indicating that the role of Mre11‐Rad50 in nucleoid reorganisation is independent of homologous recombination. We therefore propose that nucleoid compaction is part of a DNA damage response that accelerates cell recovery by helping DNA repair proteins to locate their targets, and facilitating the search for intact DNA sequences during homologous recombination.