DNA Repair 3 (2004) 845–854 Review The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together Travis H. Stracker1 , Jan-Willem F. Theunissen1 , Monica Morales, John H.J. Petrini∗ Molecular Biology Program, Memorial Sloan Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY 10021, USA Available online 9 April 2004 Abstract The conserved Mre11 complex (Mre11, Rad50, and Nbs1) plays a role in each aspect of chromosome break metabolism. The complex acts as a break sensor and functions in the activation and propagation of signaling pathways that govern cell cycle checkpoint functions in response to DNA damage. In addition, the Mre11 complex influences recombinational DNA repair through promoting recombination between sister chromatids. The Mre11 complex is required for mammalian cell viability but hypomorphic mutants of Mre11 and Nbs1 have been identified in human genetic instability disorders. These hypomorphic mutations, as well as those identified in yeast, have provided a benchmark for establishing mouse models of Mre11 complex deficiency. In addition to consideration of Mre11 complex functions in human cells and yeast, this review will discuss the characterization of mouse models and insight gleaned from those models regarding the metabolism of chromosome breaks. The current picture of break metabolism supports a central role for the Mre11 complex at the interface of chromosome stability and the regulation of cell growth. Further genetic analysis of the Mre11 complex will be an invaluable tool for dissecting its function on an organismal level and determining its role in the prevention of malignancy. © 2004 Elsevier B.V. All rights reserved. Keywords: Mre11 complex; Double strand breaks; Checkpoints; ATM The metabolism of chromosome breaks requires DNA recombination, cell cycle checkpoint, and DNA break sensing functions. The execution and integration of these functions is critical for cellular survival in the face of chromosome breakage, as cell viability is reduced by mutations that diminish break sensing, repair, or break-dependent cell cycle checkpoints. Another outcome of such mutations is a reduction in the fidelity with which genetic information is transmitted from parental cells to daughter cells. This is a state defined as genomic instability. DNA double strand breaks (DSBs) are detected by sensor molecules that trigger the activation of transducing kinases [1]. These transducers then phosphorylate effector molecules to regulate signaling cascades that control cell cycle checkpoints, influence DNA repair machinery, or trigger apoptotic pathways. An additional class of molecules termed mediators, typified by the Saccharomyces cerevisiae Rad9p, has been proposed to play an intermediate regulatory role in sig∗ Corresponding author. E-mail address: petrinij@mskcc.org (J.H.J. Petrini). 1 These authors contributed equally to this work. 1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.03.014 naling from transducer to effector molecules. The primary transducers of the DSB response are the ataxia-telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) kinases [1,2]. Mutations in the gene for ATM cause the human disease ataxia-telangiectasia (A-T) that is characterized by the loss of DSB induced checkpoint arrest, genomic instability, neurologic and immunological dysfunction, and cancer predisposition (Table 1) [2]. The Mre11 complex is a highly conserved protein complex that influences each aspect of chromosome break metabolism [3,4]. The approximately 1.5 mDa complex contains three core proteins, Mre11, Rad50, and Nbs1 (Xrs2 in S. cerevisiae) [5–8]. A description of the individual Mre11 complex components is presented in Fig. 1. Please see the legend for details. In vitro analyses with human and yeast proteins indicate that the complex specifies both 3 -5 exonuclease and single strand endonuclease activities, as well as limited DNA unwinding activity [5,8–11]. The precise functions of Mre11 complex nuclease activities in vivo have not been established, possibly due to redundancy with other cellular nucleases. Hypomorphic alleles of yeast MRE11 and RAD50 that 846 T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 Table 1 Human genome instability syndromes NBS A-TLD A-T Two alleles (N117S and R633X) >50 allelesc Number of mutant alleles Nine alleles; ∼82% Cellular phenotypes IR sensitivity MMC sensitivity Mre11 complex mislocalization Mre11 complex protein levels G1 /S checkpoint failure Intra-S checkpoint failuref G2 /M checkpoint failure IR induced Chk2 phosphorylation defect IR induced p53 stabilization defect Chromosome instability


Nbs1p70 reducedd Ce + Cg,h
h Ce



All three reduced ND + ND
i –

– – –
++



Clinical phenotypes Neuronal abnormality Neuronal degeneration Cancer predisposition Immunodeficiency Telangiectasia
–

– –
NA – – –



657del5a,b Data from [2,3,68]. Exceptions referenced separately. (
): Presence of phenotype; (–): absence of phenotype; (+): number of + signs indicate relative severity of phenotype; C: controversial; ND: not determined; NA: not applicable; only four probands reported to date. a [69,70]; % represents frequency of 657 del5 allele in NBS patients reported in these two references. b [71]: Atypical Fanconi anemia patient with NBS1 mutation (Y363X). c [72]. d [50]. e [73–75]. f [30]. g [22,76]. h [77]. i [23]. diminish the complex’s nuclease activities lead to defects in processing Spo11 induced DSBs during meiosis but confer relatively mild clastogen sensitivity and mitotic recombination defects [10,12,13]. These data argue that the complex’s influence on mitotic recombination is not significantly dependent upon its nuclease functions. Nevertheless, overproduction of Exo1, a 5 -3 exonuclease, reduces the clastogen sensitivity of Mre11 complex deficient cells [14–16], raising the possibility that the complex recruits Exo1, and potentially other enzymes to sites of DNA damage. Nuclease deficient alleles of MRE11, as well as a class of hypomorphic RAD50 alleles, called ‘S’ alleles, are lethal in strains lacking Rad27, a nuclease required for processing of Okazaki fragments, underscoring the link between Mre11 complex activity and DNA replication [17]. In mammalian cells, the Mre11 complex’s association with DSBs early in the DNA damage response is well established [18]. This property, together with the observation that the complex’s DSB association is correlated with checkpoint activation, establish that the Mre11 complex functions as a DSB sensor that collaborates with transducing kinases to impose checkpoint regulation in response to chromosome breaks. The discovery that hypomorphic mutations in the genes encoding Mre11 and Nbs1 underlie rare genetic instabil- ity disorders, ataxia-telangiectasia like disease (A-TLD) and Nijmegen breakage syndrome (NBS), provided the first indication that the Mre11 complex functioned in checkpoint signaling pathways governed by ATM (Table 1) [6,19]. ATM is the primary transducer of DSB induced checkpoint activation, evident by the failure of human or murine cells deficient in ATM to arrest at the G1/S and G2/M checkpoints after DNA damage [2]. In addition, these cells exhibit radio-resistant DNA synthesis (RDS), a manifestation of intra-S phase checkpoint dysfunction. This failure to transiently suppress DNA replication in response to damage or blockage of replication fork progression is thought to play a major role in the development of genomic instability [20]. Autophosphorylation and activation of ATM kinase activity is one of the earliest characterized events in the response to DSBs [21]. Autophosphorylation of ATM at serine-1981 leads to dimer dissociaton and it has been proposed that this leads directly to the release of active ATM monomers that modify downstream effector molecules such as Nbs1, 53BP1, Chk1, Chk2, H2AX, NFBD1/MDC1, and BRCA1 [2,21]. Although the initiating events for activation remain unclear, ATM autophosphorylation in response to IR is profoundly impaired in human Mre11ATLD1 -expressing cells, that contain a C-terminally truncated Mre11, or cells expressing adenoviral proteins that degrade the Mre11 complex T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 847 Fig. 1. The Mre11 complex. (A) Mre11 contains four conserved N-terminal phosphoesterase motifs (orange). In vitro studies have demonstrated that Mre11 has 3 -5 dsDNA exonuclease and ssDNA and dsDNA endonuclease activity (reviewed in [83]). The N117S and R633STOP mutations identified in A-TLD patients are indicated (green) [19]. The C-terminus of the protein contains two DNA binding domains (blue). (B) The wild type Nbs1p95 protein contains both an FHA (red) and BRCT (yellow) domain in the N-terminus [84]. Two phosphorylation sites for Atm at serines 278 and 343 are indicated [27–29]. The Mre11 interaction domain is located in the C-terminus [85]. Lymphoblasts from NBS patients expressing the 657del5 allele were found to contain two Nbs1 species [50]. The Nbs1p26 protein contains the FHA and BRCT domains. The Nbs1p70 protein is expressed through an alternative translation site. The N-terminus of Nbs1p70 contains 10–18 amino acids from an alternate reading frame and becomes wild type at exon 6. This protein is not detected in many NBS patient fibroblast lines. (C) The Rad50 protein contains Walker A and B motifs that confer ATPase activity to the protein (reviewed in [83]). The K22M mutation, modeled in Rad50S/S mice, is located in the Walker A box [66]. (D) The heptad repeats of the Rad50 protein interact in an intramolecular fashion bringing the Walker A and B motifs into proximity [86]. The central portion of the protein forms a zinc hook that may facilitate interactions between Rad50 molecules [38]. (E) The Rad50 hook domain. A hypothetical disposition of Mre11 complex dimers linking sister chromatids is shown. Cysteine residues within the Rad50 hook domain coordinate a zinc atom (inset), linking Mre11 complex monomers distal to the DNA binding domain(s) at the globular domain. The globular domain contains the Walker A (gold) and B (copper) ATP binding motifs, Mre11 (silver) and Nbs1 (not shown). The geometry of zinc coordination orients the coiled coil arms of Rad50 monomers outward as shown. Evidence of flexibility at particular regions (not shown) of the arms has been established [87]. Hence, the intersister distance is likely to be less than suggested by this representation. [22,23]. Extensive cytological analysis of the Mre11 complex establish that its association with chromatin or damaged DNA is independent of ATM, and is thus physically upstream of ATM with respect to break detection [24,25]. Collectively, these data strongly support that the Mre11 complex is a sensor that signals to ATM from chromatin [18]. It remains to be determined whether all members of the Mre11 complex are required and the precise mechanism by which the complex triggers ATM activation. In addition to functioning as a sensor, the Mre11 complex plays a crucial role as an ATM effector. Serine-343 of Nbs1 was identified as a critical ATM target for activation of the intra-S phase checkpoint [26–29]. Cells from NBS and A-TLD patients display intermediate RDS phenotypes compared to A-T cells indicating that ATM activation is at least partially retained in those mutants [30]. ATM phosphorylates and activates Chk2 in response to DSBs [31]. Full Chk2 phosphorylation after damage requires the Mre11 complex, which may bring Chk2 in contact with ATM at chromosome breaks [32]. Subsequent to Chk2 activation, Chk2 phosphorylates Cdc25a leading to inhibition of Cdk2, thus blocking the initiation of DNA replication [30,33]. In parallel, phosphorylated Nbs1 functions in a pathway involving NFBD1/MDC1, SMC1, and possibly other unidentified proteins [34–36]. Together these pathways control DNA synthesis and maintain genomic stability during S-phase. The Mre11 complex also exerts a profound influence on DNA recombination, primarily in homology directed repair of DSBs between sister chromatids. Insight regarding the mechanistic basis for the complex’s effect on intersister recombinational DNA repair has come from structural characterization of the internal coiled coil domain of Rad50. Scanning force microscopy reveals that complexes of human Mre11 and Rad50 bind DNA via a globular domain, while the coiled coil regions of Rad50 form an extended intramolecular flexible arm. Thus, interactions between the 848 T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 that establishment of chromatin association during replication reflects the Mre11 complex’s role in promoting sister chromatid association and recombination. Further, there is a perfect correlation between attenuated ␥-irradiation-induced chromatin association (e.g., in NBS and A-TLD) and cell cycle checkpoint deficiency. Hence, these otherwise diverse influences on DSB metabolism are governed by a common event, the Mre11 complex’s association with chromatin. 1. Lessons from animal models of Mre11 complex hypomorphism Fig. 2. The murine Nbs1p80 protein is expressed in a mouse model of NBS [42]. (A) The schematic diagram shows the similarity between the human Nbs1p70 and murine Nbs1p80 proteins. The murine Nbs1p80 protein lacks the conserved FHA and BRCT domains but retains the ATM consensus phosphorylation sites and the Mre11 interaction domain. (B) An alignment of the murine Nbs1p80 and human Nbs1p70 amino acid sequences is shown (from [42]). Unique sequence is indicated in blue and arises from alternative translation or from the targeting construct in the case of murine Nbs1p80 . The wild type sequence of exon 6 is indicated by boxed text. coiled-coils of DNA bound Mre11 complexes would tether sister chromatids or DNA ends within the same chromatid [37]. A conserved ‘hook’ motif within the coiled coil domain of Rad50 appears to account for interaction between coiled coil arms [38]. This motif contains two conserved cysteine residues, which when complexed with a second hook region, coordinate a zinc atom and form an interlocking hook with the intramolecular coiled coil Rad50 arms extending outward in opposite polarities (Fig. 2). The hook region is clearly a major determinant of Mre11 complex assembly and function. Mutation of the Zinc-coordinating Cys residues in S. cerevisiae Rad50 to Ser abolished Mre11 interaction, demonstrating the importance of the hook for the overall integrity of the complex. A Cys to Gly allele imparted radiation sensitivity and slow growth, supporting the hypothesis that the hook may be relevant to DNA repair and replication [38]. These structural data provide an as yet speculative, but compelling molecular basis for the Mre11 complex to facilitate sister chromatid interaction. Chromatin association, whether at sites of DNA damage or established during DNA replication appears to be a prerequisite for both the DNA recombination and cell cycle checkpoint functions of the complex. We have proposed In contrast to S. cerevisiae, the assessment of mammalian Mre11 complex functions in vivo is complicated by the inviability of null Mre11, Rad50, and Nbs1 mutants in cultured cells and in vivo [39–41]. Hypomorphic Nbs1 and Mre11 alleles in the rare human syndromes NBS and A-TLD provided a benchmark for establishing viable Nbs1 and Mre11 mutants in the mouse [6,19,42,43] (Table 2). Rad50 mutations in humans have not been reported; however, a class of RAD50 alleles in S. cerevisiae, rad50S alleles, offered an ideal means to address these issues regarding the physiological role of the Mre11 complex: its role in meiosis, in DNA replication, and in DNA damage sensing and signaling. The relevant phenotypic features of the S. cerevisiae rad50S mutants are discussed below. The Rad50S allele Rad50k22M (rad50-R20M in S. cerevisiae) was modeled in mice and has provided important insights. In the following sections, we summarize the information garnered from genetic analyses in mice. Together with evidence from S. cerevisiae and human cells, the data in hand collectively support the following interpretations: • The Mre11 complex functions as a DNA damage sensor. • The Mre11 complex’s influence on DNA damage checkpoints is not exclusively associated with ATM function. • The Mre11 complex’s meiotic functions in S. cerevisiae do not appear to be directly recapitulated in mammals. • Intra S and G2/M checkpoints defects enhance the penetrance of cancer predisposing recessive mutations, but are insufficient for the initiation of malignancy, in spite of the chromosome instability associated with such defects. 2. Mouse models of NBS and A-TLD Mouse models of Mre11 complex deficiency have been generated by mimicking the mutations identified in human NBS and A-TLD patients. Two mouse models harboring N-terminally truncated forms of Nbs1, Nbs1 B / B and Nbs1m/m , have been developed to study Nbs1 functions at the organismal level (Fig. 2) [42,44]. More recently we have generated a murine model of A-TLD, Mre11ATLD1/ATLD1 , harboring a hypomorphic mutation in Mre11 [43]. Phenotypic analysis of these mice has confirmed that the Mre11 T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 849 Table 2 The Mre11 complex mouse models Cellular phenotypes IR sensitivity Mre11 complex mislocalization Mre11 complex protein levels G1 /S checkpoint failure Intra-S checkpoint failure G2 /M checkpoint failure Chk2 phosphorylation defect p53 stabilization defect Chromosome instabilityb Lack of IR-dependent thymocyte apoptosis Organismal phenotypes Embryonic death Survival (in months) Survival in p53−/− background (in months)d Survival in p53+/− background (in months)f B and T cell development T cell receptor transrearrangements Germ cells RAD50S /S Nbs1 – – WT

Nbs1 – – – – – – – 40% Rad50S /S die prenatally 2.6 3.3 5.0 Hematopoietic stem cell failure – Fertile, male germ cell attrition B/ B Mre11ATLD1/ATLD1 Atm−/−
– WT +a +

– + +

Mre11ATLD1 , Rad50, and Nbs1 reduced + +

– + + Maternal, blastocyst stage WT 2.8 11.2 WT Maternal, blastocyst stage WT 3.7 7.7g WT –c
Female subfertile
Female subfertile B reduced ++ ++


++ ++ 5 2e ND T cell development failureh
Infertile All data is from [42,43,66], except where indicated. (
): Presence of phenotype; (–): absence of phenotype; (+): number of + signs indicate relative severity of phenotype; ND: not determined. a [52]. b Karyotypic analysis on primary MEFs, except on ear fibroblasts for Rad50S /S . c 60% of Atm−/− p53−/− mice die prenatally [78]. In C57BL6/J inbred strain, 16.3% Atm−/− instead of the expected 25% [79]. d Compared to average age of 4.5 months for p53−/− mice. e [78]. f Compared to average age of 18.4 months for P53+/− mice. g Based on five Mre11ATLD1/ATLD1 p53+/− mice. h [80–82]. complex plays a central role in ATM mediated signaling pathways, and has also indicated that the complex mediates functions outside of ATM-dependent DNA damage responses that are essential for cellular viability, as predicted from analysis of Mre11 complex deficiency in S. cerevisiae [45]. Nbs1 B / B mice contain an N-terminally truncated protein, Nbs1p80 , that lacks the forkhead associated (FHA) and BRCA1 C-terminal (BRCT) domains which, as a general class, mediate phosphorylation dependent protein interactions [42,46–48]. Specific Nbs1 FHA domain interactions with NFBD1/MDC1 and ␥-H2AX have been suggested [34,49], but the purported Nbs1-␥-H2AX interaction remains to be clearly established. Nbs1p80 is similar to the Nbs1p70 protein present in human NBS cells, the difference in nomenclature is historical rather than descriptive [50]. The Nbs1 B allele encoding Nbs1p80 has been sequenced and the amino acid sequence has been verified using antibodies to the unique N-terminus [42]. Nbs1p80 protein is present at low levels in Nbs1 B / B MEFs and, unlike Mre11 mutants, Nbs1 B / B MEFs do not exhibit a reduction in the levels of Mre11 and Rad50 [51]. The interaction between Mre11 and Nbs1p80 remains intact [42]. As in NBS patient cells, cells from Nbs1 B / B animals exhibited mislocalized Mre11; in Nbs1 B / B MEFs, Mre11 is more evenly distributed between the nucleus and cytoplasm and the predominantly nuclear localization observed in wild type (WT) cells is lost [42]. Absent a more detailed characterization of the Nbs1m allele, and the protein it encodes, reconciliation of several phenotypic differences with Nbs1 B / B is precluded [44]. Mre11ATLD1/ATLD1 mice express one of the two A-TLD mutations identified in humans [19,43]. This mutation results in a decrease in the protein levels of all three complex members, although the complex remains physically intact. The reduced protein levels in Mre11ATLD1/ATLD1 precluded robust immunofluorescence analysis, but the normally restricted nuclear localization of the complex as well as its ability to form IR induced foci is compromised [43]. 3. Cell cycle checkpoint defects Cell lines from Mre11ATLD1/ATLD1 , Nbs1 B / B , and Nbs1m/m mice recapitulate many aspects of Mre11 850 T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 complex functional hypomorphism observed in cells from A-TLD and NBS patients, including checkpoint dysfunction and sensitivity to a variety of clastogens [6,19,42–44]. Mre11ATLD1/ATLD1 and Nbs1 B / B MEFs exhibit a milder G1/S checkpoint defect than Atm−/− cells and show normal stabilization of both p53 and p21 levels after ionizing radiation (Table 2) [42,43,52]. This suggests that the complex may affect only a subset of ATM mediated phosphorylation events or that its sensor functions are not operative in G1. In Mre11ATLD1/ATLD , Nbs1 B / B , and Nbs1m/m cells, as in their human counterparts, the degree of RDS appears to be intermediate between wild type and Atm−/− [30]. G2/M checkpoint defects are observed in Nbs1 B / B and Mre11ATLD1/ATLD1 cells, and as with RDS, the defect is less severe than in Atm−/− cells (Table 2); the ATM dependent G2/M checkpoint has not been assessed in Nbs1m/m [42,44]. The role of the Mre11 complex in ATM/ATR mediated administration of the G2/M checkpoint has not been well characterized but ultimately it is likely to reflect impaired ATM/ATR signaling to the checkpoint kinase Chk1 [53]. Consistent with this view, evidence for Nbs1 dependent phosphorylation of Chk1 has been established [36,54]. between Nbs1m/m and Mre11ATLD1/ATLD1 or Nbs1 B / B remains unresolved. The phenotypes of Nbs1 B / B and Mre11ATLD1/ATLD1 mice clearly establish that chromosome instability is insufficient to enhance the intiation of malignancy, whereas it strongly potentiates the penetrance of initiating lesions, as exemplified in p53+/− mice. Several lines of evidence indicate that selection for mutations that abrogate apoptotic pathways is critical to lymphomagenesis in the mouse [56,57]. The failure of Mre11ATLD1/ATLD1 and Nbs1 B / B to enhance lymphomagenesis in p53−/− mice reflects the fact that p53 surveillance is inoperative when chromosome breakage in Nbs1 B / B and Mre11ATLD1/ATLD1 occurs. In either the single or double mutants, cells harboring broken chromosomes at metaphase are likely to be inviable due to segmental aneuploidy [58,59]. Hence, these lesions would not synergize with p53 deficiency. In p53 proficient Mre11ATLD1/ATLD1 and Nbs1 B / B mutants, chromosome breaks arising in S-phase would not signal to p53, and thus not impose strong selection for abrogation of p53 mediated apoptosis. 5. Embryonic viability 4. Chromosome instability and tumorigenesis Cells from both Mre11ATLD1/ATLD1 and Nbs1 B / B mice exhibit chromosome instability, defined in this context as breakage and rearrangement; changes in ploidy are not observed [42,43]. Chromosome instability in Atm−/− MEFs is increased approximately twofold compared to Mre11ATLD1/ATLD1 and this difference may reflect the relative severity of the checkpoint defects in these cell lines (Table 2). Surprisingly, Mre11ATLD1/ATLD1 and Nbs1 B / B mice are not prone to malignancy, in spite of the associated checkpoint defects and chromosomal instability [42,43]. Further, these mutations have no effect on tumor latency in p53−/− mice. In contrast to p53−/− deficiency, Mre11ATLD1/ATLD1 and Nbs1 B / B have a severe impact on tumor latency in p53+/− animals (Table 2) [43]. Because checkpoint deficiency in S. cerevisiae is strongly associated with elevated mitotic recombination and chromosomal rearrangement [55], we have proposed that the reduced tumor latency in Mre11ATLD1/ATLD1 p53+/− and Nbs1 B / B p53+/− mice reflects an increase in the frequency of loss of heterozygosity (LOH) at the p53 locus. The frequent occurrence of chromatid gaps and breaks in Mre11ATLD1/ATLD1 and Nbs1 B / B cells offer an appealing mechanistic basis for increased LOH, as these lesions would be highly recombinogenic. Nbs1m/m animals exhibit a similar spectrum of checkpoint defects as Mre11ATLD1/ATLD1 and Nbs1 B / B , but are predisposed to lymphomagenesis and exhibit immunodeficiency [44]. The striking difference in cancer predisposition The Mre11ATLD1/ATLD1 mice exhibit a profound maternal defect in embryonic viability that is evident to a lesser extent in Nbs1 B / B (Table 2) [43]. In Mre11ATLD1/ATLD1 females, embryos show a frequent, but stochastic failure in proliferation 5–10 cell divisions after fertilization. The rare embryos that develop to term in Mre11ATLD1/ATLD1 females are normal. The maternal effect on embryonic viability could not be explained by a defect in female meiosis. Germinal vesicle breakdown (GVBD) oocytes contain twenty bivalents held together by chiasmata, and meiosis II arrested oocytes exhibit no aneuploidy. We submit that the death of embryos is likely to result from chromosome breakage, analogizing the situation proposed for Mre11ATLD1/ATLD1 or Nbs1 B / B lymphocytes which die as a result of segmental aneuploidy. However, in embryos, unlike cells from the adult animal, p53-independent apoptotic pathways operative in the embryonic stages may also precipitate cellular attrition. 6. Meiosis Oocyte development in Mre11ATLD1/ATLD1 females is apparently normal, in sharp contrast to sporulation in yeast mutants with C-terminal Mre11 truncations, where spores are generally inviable [8,60,61]. Deletion of as little as 49 amino acid residues (mre11 C49 ; [61]) or as many as 137 amino acid residues from the C-terminus (mre11-5; [8]) eliminates meiotic recombination, and leads to the production of achiasmate, inviable spores. Although Mre11ATLD1 T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 encodes a 75 amino acid C-terminal deletion, it does not recapitulate the meiotic phenotypes of C-terminally truncated S. cerevisiae mutants. Similarly, the murine Rad50S allele, which corresponds to the S. cerevisiae rad50-R20M allele, failed to recapitulate the meiotic phenotype observed in S. cerevisiae. In that, and other S. cerevisiae rad50S mutants, meiotic DSBs are not processed due to covalent attachment of Spo11 at the site of the DSB that initiates meiotic recombination [62]. These data support the surprising conclusion that, despite the extraordinary conservation of Mre11 and Rad50, the meiotic requirements for Mre11 complex functions may differ in mammals and yeast. 7. Rad50 mutants S. cerevisiae rad50S mutants exhibit a number of phenotypic features that we reasoned would provide novel insights in the mouse. First, S. cerevisiae rad50S mutants are blocked early in meiotic progression. This reflects the yeast Mre11 complex’s role in cooperating with the topoisomerase II like enzyme, Spo11, which is responsible for creating the DSBs that initiate meiotic recombination [63]. Second, rad50S mutations are synthetically lethal with deficiency in Rad27, the nuclease responsible for processing Okazaki fragments [15,64]. This indicates that the rad50S mutation affects the metabolism of DNA replication intermediates, and suggested that it might impart chromosome instability in the mouse. Finally, despite a lack of clastogen sensitivity and wild type proficiency in DNA recombination, we observed constitutive DNA damage signaling in S. cerevisiae rad50S mutants [65]. On this basis, it appeared that the rad50S allele might represent a gain of function in the DNA damage signaling functions of the complex [18]. Unlike Rad50 / mice which were embryonic lethal, Rad50S /S mice exhibited only partial embryonic lethality (Table 2) [66]. Rad50S /S mice had a shortened lifespan associated with total hematopoietic failure due to stem cell attrition. By 2 months of age, most Rad50S /S animals developed complete aplastic anemia, and died at 3 months whereas relatively rare longer-lived animals were predisposed to malignancy (Table 2). Surprisingly, although Rad50S /S males experienced apoptotic cellular attrition in the testis, they were not overtly defective in meiotic progression, and both males and females appeared to be fertile. Thus the Rad50S allele in mice does not recapitulate the meiotic phenotype observed in the corresponding S. cerevisiae (rad50-R20M) mutant. The profound effect of the Rad50S mutation (in mice contrasts dramatically with the effects observed at the cellular level. Wild-type levels of Mre11, Nbs1 and Rad50S protein are present in MEFs, and the formation of the Mre11 complex is not affected. Rad50S /S MEFs display no growth defect, nor are they sensitive to DNA-damaging agents (Table 2). Consistent with the observation that the formation of Mre11 IR-induced foci is also unaffected, the checkpoint functions of the Mre11 complex remain intact in Rad50S /S 851 cells [66]. However, we observed indices of chronic genotoxic stress in Rad50S /S cells such as increased basal levels of ␥-H2AX and apoptosis. Although a modest increase in chromosomal instability was noted, the lack of clastogen sensitivity in Rad50S /S cells clearly indicated that the DNA repair functions of the complex were not grossly affected. This is analogous to the phenotypic features of rad50S mutants in S. cerevisiae [12], which similarly exhibit elevated indices of DNA damage signaling [65] without overt defects in chromosome break metabolism. Consistent with the interpretation that Rad50S /S phenotypes arose from chronic genotoxic stress, p53 deficiency completely mitigated the cellular attrition associated with the Rad50S mutation. Rad50S /S p53−/− mice die at approximately the same age as Rad50S /S ; however they succumb to lymphomas with an otherwise intact hematopoietic compartment (Table 2). Rad50S /S p53+/− mice are longer-lived than Rad50S /S , while the latency of tumorigenesis associated with p53+/− mice is reduced and shifted from solid tumors to lymphomas. In Nbs1 B / B and Mre11ATLD1/ATLD1 , these outcomes are likely a consequence of chromosome instability. They may be similarly attributed to the modest chromosome instability observed in Rad50S /S . An alternative and non-exclusive interpretation is that chronic DNA damage signaling by the Rad50S allele imposes strong selection for loss of p53-dependent apoptosis. S. cerevisiae rad50S suppresses Mec1 deficiency via activation of a Tel1-Mre11 pathway, and both Rad53 and Rad9, downstream mediators of the DNA damage response, are constitutively activated in unstressed cells [65]. The phenotype of Rad50S /S mice clearly indicates that constitutive DNA damage signaling, and hence constitutive selection for abrogation of apoptotic functions, occurs in Rad50S /S mice. We have proposed that the Rad50S mutation is a gain of function that results in constitutive DNA damage signaling [18]. To test this hypothesis, Rad50S /S Atm−/− mice were established. We reasoned that if the effect of Rad50S /S on p53+/− was attributed to chromosome instability, that endpoint would be more severe in the double mutants, as was observed in DNA ligase IV-deficent Atm−/− mice [67]. In contrast, the alternative interpretation of chronic signaling would predict that the Rad50S allele would mitigate the chromosome instability of Atm−/− . Supporting this latter interpretation, preliminary evidence suggests that Rad50S increases the survival and tumor latency of Atm−/− mice. 8. Synthetic lethalities: functions of the Mre11 complex outside the ATM pathway Intercrosses of Nbs1 B / B and Mre11ATLD1/ATLD1 mice have revealed some surprising synthetic lethal genetic interactions. To be sure, lethality provides very little resolving power in defining the relative dispositions of DNA damage response components. In particular, it is difficult to distinguish additivity (most consistent with impairment 852 T.H. Stracker et al. / DNA Repair 3 (2004) 845–854 of a single pathway) from synergy (most consistent with the impairment of parallel pathways). Nevertheless, certain conclusions can be drawn. Whereas Nbs1 B / B Prkdcscid /scid are viable [52], Atm−/− Prkdcscid /scid and Mre11ATLD1/ATLD1 Prkdcscid /scid mice are inviable [51]. These outcomes likely reflect that DNAPKcs functions outside the canonical non-homologous endjoining pathway [67]. And, it is likely that loss of this function synergizes with the defects ensuing from Mre11ATLD1 and Nbs1 B . Reflecting the milder phenotype of Nbs1 B , the double mutants are viable, whereas Mre11ATLD1/ATLD1 Prkdcscid /scid are not. Perhaps more surprisingly, Mre11ATLD1/ATLD1 and Nbs1 B / B mice are inviable in the context of ATM deficiency [42,51]. The inviability of Mre11ATLD1/ATLD1 Atm−/− and Nbs1 B / B Atm−/− mice underscores that the Mre11 complex functions outside the DNA damage response pathway governed by ATM. It is possible that these functions involve other signal transduction pathways, such as those governed by ATR [22,42]. An alternative possibility is that the additive toxicity of the chromosome breakage and checkpoint defects ensuing from either of the single mutations transcends the level that permits viability, that is, the defects may impair the same pathway, but are additive. Both interpretations are supported by the data to some degree, but given that the S. cerevisiae complex can signal to both Mec1 and Tel1 [65], we favor the hypothesis that ATR activation is compromised in Mre11ATLD1/ATLD1 and Nbs1 B / B , and that loss of ATM reduces the aggregate activity of the checkpoint pathways to a level that is incompatible with survival of the developing embryo. As the endpoints of both the ATM and ATR dependent pathways become clearer, this hypothesis will become testable. Moreover, a more nuanced view of ATM and ATR endpoints may well reveal that the physiological roles played by these transducers of the DNA damage response overlap to a greater extent than currently appreciated. 9. Epilogue The Mre11 complex has been extensively analyzed at the biochemical level, and important insights have been garnered from cell biological approaches as well. It could be said that as many questions as answers regarding the in vivo roles of the complex, and the mechanisms by which they are mediated, have arisen from genetic approaches. However, the availability of mutant strains and the information obtained in their phenotypic characterization will constitute an important resource for solving the abiding mysteries. Primary among them is the mechanism by which the complex influences cell cycle checkpoint during S phase and at the G2/M boundary. In addition, although the complex’s nuclease functions are highly conserved, they do not appear to be a primary determinant of Mre11 complex DNA recombination func- tions. We have proposed that the complex’s influence on recombination is structural rather than enzymatic, and further that this structural role is mediated by the Rad50 hook domain which physically bridges the participants in the DNA recombination process. 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