Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity

DNA double-strand breaks (DSBs) can be processed by the Mre11-Rad50-Nbs1 (MRN) complex, which is essential to promote ataxia telangiectasia-mutated (ATM) activation. However, the molecular mechanisms linking MRN activity to ATM are not fully understood. Here, using Xenopus laevis egg extract we show that MRN-dependent processing of DSBs leads to the accumulation of short single-stranded DNA oligonucleotides (ssDNA oligos). The MRN complex isolated from the extract containing DSBs is bound to ssDNA oligos and stimulates ATM activity. Elimination of ssDNA oligos results in rapid extinction of ATM activity. Significantly, ssDNA oligos can be isolated from human cells damaged with ionizing radiation and injection of small synthetic ssDNA oligos into undamaged cells also induces ATM activation. These results suggest that MRN-dependent generation of ssDNA oligos, which constitute a unique signal of ongoing DSB repair not encountered in normal DNA metabolism, stimulates ATM activity.     

DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA

The Mre11–Rad50–Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.     

ATP‐dependent DNA binding,unwinding, and resection by the Mre11/Rad50 complex

ATP‐dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double‐strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here, Methanococcus jannaschii MR‐ATPγS‐DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS‐bound Rad50 nucleotide‐binding domains. Duplex DNA cannot access the Mre11 active site in the ATP‐free full‐length MR complex. ATP hydrolysis drives rotation of the nucleotide‐binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis‐driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5' end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis.     

Structure of the Rad50 DNA double‐strand break repair protein in complex with DNA

The Mre11–Rad50 nuclease–ATPase is an evolutionarily conserved multifunctional DNA double‐strand break (DSB) repair factor. Mre11–Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50^NBD (nucleotide‐binding domain) in complex with Mre11^HLH (helix‐loop‐helix domain), AMPPNP, and double‐stranded DNA. DNA binds between both coiled‐coil domains of the Rad50 dimer with main interactions to a strand‐loop‐helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double‐strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP‐bound state.     

Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2

In this study, we investigate the interplay between Ku, a central non‐homologous end‐joining component, and the Mre11–Rad50–Xrs2 (MRX) complex and Sae2, end‐processing factors crucial for initiating 5′‐3′ resection of double‐strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Δ sgs1Δ) cannot be suppressed by the yku70Δ mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication‐associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end‐processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.     

Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M _r 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca²⁺ channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M _r 32 000 required protein phosphatase 1/2A activity and extracellular Ca²⁺. In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na⁺ or Ca²⁺ sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.     

Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.     

Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

Single‐stranded DNA constitutes an important early intermediate for homologous recombination and damage‐induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double‐strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5′‐strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2‐dependent initiation to the Exo1‐ and Dna2‐dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.     

Human DNA damage response and repair deficiency syndromes: Linking genomic instability and cell cycle checkpoint proficiency

A plethora of clinically distinct human disorders exist whose underlying cause is a defect in the response to or repair of DNA damage. The clinical spectrum of these conditions provides evidence for the role of the DNA damage response (DDR) in mediating diverse processes such as genomic stability, immune system function and normal human development. Cell lines from these disorders provide a valuable resource to help dissect the consequences of compromised DDR at the molecular level. Here we will discuss some well known, less well known and ‘novel’ DDR defective disorders with particular reference to the functional interplay between the DNA damage response and cell cycle checkpoints. We will describe recent advances in further delineating the genetic basis of Seckel syndrome and microcephalic osteodysplastic primordial dwarfism type II, which have shed more light on the interplay between the DDR, cycle progression and centrosomes. We will also overview recent developments concerning haploinsufficiency of DDR components and their association with certain genomic disorders such as Miller–Dieker lissencephaly syndrome and Williams–Beuren syndrome. Finally, we will discuss how defects in the DDR result in some unexpected clinical features before describing how the nature of a DDR defect impacts on the management and treatment of individuals with these conditions.     

Rad50S alleles of the Mre11 complex: questions answered and questions raised

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; D'D. Amours, S.P. Jackson, The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15 (2001) 2238-49. ; M. Grenon, C. Gilbert, N.F. Lowndes, Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3 (2001) 844-847. ] where the signaling is compromised. Herein, we describe evidence for chronic signaling by Rad50S and discuss possible mechanisms.     

Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis

Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.     

Interplay of two major repair pathways in the processing of complex double-strand DNA breaks

Radiation-induced complex double-strand breaks (DSBs) characterised by base lesions, abasic sites or single-strand breaks in close proximity to the break termini, are believed to be a major cause of the biological effects of ionising radiation exposure. It has been hypothesised that complex DSBs pose problems for the repair machinery of the cell. Using a biochemical approach, we have investigated the challenge to two major repair processes: base excision repair and ligation of DSB ends. Double-stranded oligonucleotides were synthesised with 8-oxo-7,8-dihydroguanine (8-oxoG) at defined positions relative to readily ligatable 3'-hydroxy or 5'-phosphate termini. The break termini interfere with removal of 8-oxoG during base excision repair as elucidated from the severely reduced efficiency of 8-oxoG removal by OGG1 with AP endonuclease-1 when in close proximity to break termini. NEIL-1, however, can partially restore processing of complex DSBs in an AP endonuclease-1 independent manner. The influence of 8-oxoG on ligation shows delayed rejoining if 8-oxoG is positioned two to three bases from the 3'-hydroxy or six bases from the 5'-phosphate termini. When two 8-oxoG lesions are positioned across the break junction ligation is severely retarded. This reduced efficiency of repair indicates that complex DSBs are likely to persist longer than simple DSBs in cells, and as a consequence are more significant in contributing to the biological effects of ionising radiation.     

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.