Artemis and Nonhomologous End Joining-Independent Influence of DNA-Dependent Protein Kinase Catalytic Subunit on Chromosome Stability

Deficiency in both ATM and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is synthetically lethal in developing mouse embryos. Using mice that phenocopy diverse aspects of Atm deficiency, we have analyzed the genetic requirements for embryonic lethality in the absence of functional DNA-PKcs. Similar to the loss of ATM, hypomorphic mutations of Mre11 (Mre11^ATLD1) led to synthetic lethality when juxtaposed with DNA-PKcs deficiency (Prkdc^scid). In contrast, the more moderate DNA double-strand break response defects associated with the Nbs1^ΔB allele permitted viability of some Nbs1^ΔB/ΔB Prkdc^scid/scid embryos. Cell cultures from Nbs1^ΔB/ΔB Prkdc^scid/scid embryos displayed severe defects, including premature senescence, mitotic aberrations, sensitivity to ionizing radiation, altered checkpoint responses, and increased chromosome instability. The known functions of DNA-PKcs in the regulation of Artemis nuclease activity or nonhomologous end joining-mediated repair do not appear to underlie the severe genetic interaction. Our results reveal a role for DNA-PKcs in the maintenance of S/G₂-phase chromosome stability and in the induction of cell cycle checkpoint responses.The Mre11 complex, consisting of Mre11, Rad50, and Nbs1 (Xrs2 in Saccharomyces cerevisiae), is involved in diverse aspects of DNA double-strand break (DSB) metabolism. The Mre11 complex acts as a DSB sensor, mediates cell cycle checkpoint arrest and apoptosis, and promotes DSB repair (47, 48). The influence of the Mre11 complex on DSB responses is attributable partly to its influence on ataxia-telangiectasia mutated (ATM) kinase activity (29). ATM is a central signal transducer in the response to DSBs and is required for arrest throughout the cell cycle, as well as the efficient execution of apoptosis in response to many types of genotoxic stress (43).The Mre11 complex is required for ATM activation and governs the phosphorylation of ATM substrates such as SMC1, Chk2, and BID (4, 6, 26, 47, 49, 51). The C terminus of Nbs1 interacts with ATM and plays an important role in facilitating a subset of these events, particularly those important for apoptosis (11, 14, 47, 58). However, ATM makes multiple functional contacts with members of the Mre11 complex. Nbs1, Mre11, and Rad50 are all ATM substrates, and many aspects of ATM checkpoint signaling are impaired by hypomorphic Mre11 and Nbs1 mutations that do not affect the ATM binding domain in the C terminus of Nbs1 (32, 36, 52, 54).Several molecular and genetic observations support the view that the Mre11 complex''s role in preserving genome stability is particularly relevant to the S and G₂ phases of the cell cycle (3, 56). The complex, predominantly nucleoplasmic in G₁ cells, becomes predominantly chromatin associated and colocalizes with PCNA throughout S phase (35, 38). This association is a likely prerequisite for the complex''s influence on DNA damage signaling as well as DNA repair.Cell cultures established with samples from patients with Nijmegen breakage syndrome (NBS1 hypomorphism) and ataxia-telangiectasia-like disorder (MRE11 hypomorphism) exhibit checkpoint defects in S phase and at the G₂/M transition, while the G₁/S transition is relatively unaffected. These checkpoint defects are correlated with reduced Mre11 complex chromatin association both in human cells and in mouse models of Nijmegen breakage syndrome and ataxia-telangiectasia-like disorder (5, 45, 49, 52). Chromosomal aberrations arising in these cells are predominantly chromatid type breaks, consistent with impaired metabolism of DNA replication-associated DNA breaks (49, 52).Further supporting a predominant role for the Mre11 complex in S phase is the observation that its primary role in DSB repair is the promotion of recombination between sister chromatids (3, 24). Structural and genetic evidence that the Mre11 complex effects molecular bridging between DNA duplexes offers a mechanistic basis for this observation (10, 23, 53). Molecular bridging by the Mre11 complex may also contribute to its influence on nonhomologous end joining (NHEJ) (12, 34, 57). Collectively, these data strongly support the view that the Mre11 complex''s checkpoint and DSB repair functions are manifested predominantly in the S and G₂ phases of the cell cycle.Although the Mre11 complex and ATM function in the same arm of the DNA damage response, ATM deficiency is lethal in hypomorphic Mre11 and Nbs1 mutants (Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB mice, respectively) (49, 52), suggesting that aspects of ATM function are Mre11 complex independent. ATM deficiency is also synthetically lethal with mutations in Prkdc, the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is mutated in mice with severe combined immunodeficiency (Prkdc^scid mice) (22, 42). DNA-PKcs is an ATM paralog required for NHEJ, which appears to be the predominant mode of DSB repair in G₁ cells (16).Defective NHEJ is unlikely to be the basis for the embryonic lethality of Prkdc^−/− Atm^−/− or Prkdc^scid/scid Atm^−/− mice, as loss of ATM rescues the late embryonic lethality of both DNA ligase IV (Lig4) and XRCC4 null embryos, which have more severe NHEJ defects than Prkdc^scid mice abolished by the Atm^−/− genotype (31, 42). These observations argue that the DNA-PKcs functions required for viability in the absence of ATM do not include NHEJ.To address this issue, we crossed Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB mice with Prkdc^scid/scid mice. As these Mre11 complex hypomorphs do not completely phenocopy ATM deficiency, we reasoned that double-mutant animals would be viable and thus provide a venue in which to examine the functional relationship between the Mre11 complex/ATM arm of the DNA damage response and DNA-PKcs. Whereas the Mre11^ATLD1/ATLD1 mutation was synthetically lethal with the Prkdc^scid/scid genotype, some Nbs1^ΔB/ΔB Prkdc^scid/scid mice were born, consistent with the more moderate DNA damage response defects associated with the Nbs1^ΔB allele than with the Mre11^ATLD1 allele (48). Nbs1^ΔB/ΔB Prkdc^scid/scid embryos were born at drastically reduced Mendelian ratios, displayed gross developmental defects, and were severely runted. Nbs1^ΔB/ΔB Prkdc^scid/scid cell cultures exhibited profound chromosome instability, growth defects, and increased sensitivity to ionizing radiation (IR). DNA repair defects associated with DNA-PKcs deficiency did not appear to underlie the observed phenotypic synergy. Rather, the data suggest a novel regulatory function of DNA-PKcs in the maintenance of chromosomal stability during the S and G₂ phases of the cell cycle.     

Kinase-Independent Functions of TEL1 in Telomere Maintenance

TEL1 is important in Saccharomyces cerevisiae telomere maintenance, and its kinase activity is required. Tel1p associates with telomeres in vivo, is enriched at short telomeres, and enhances the binding of telomerase components to short telomeres. However, it is unclear how the kinase activity and telomere association contribute to Tel1p''s overall function in telomere length maintenance. To investigate this question, we generated a set of single point mutants and a double point mutant (tel1^KD) of Tel1p that were kinase deficient and two Xrs2p mutants that failed to bind Tel1p. Using these separation-of-function alleles in a de novo telomere elongation assay, we found, surprisingly, that the tel1^KD allele and xrs2 C-terminal mutants were both partially functional. Combining the tel1^KD and xrs2 C-terminal mutants had an additive effect and resembled the TEL1 null (tel1Δ) phenotype. These data indicate that Tel1p has two separate functions in telomere maintenance and that the Xrs2p-dependent recruitment of Tel1p to telomeres plays an important role even in the absence of its kinase activity.The telomere is a highly ordered complex of proteins and DNA found at the ends of linear chromosomes that functions to protect the ends and prevents them from being recognized as double-strand DNA breaks (51). Telomeres shorten gradually due to incomplete replication (1, 20), and this shortening is counteracted by telomerase, which elongates telomeres (18, 19).Saccharomyces cerevisiae telomeres are composed of 300 ± 50 bp of the sequence TG_1-3/C_1-3A. The yeast telomerase complex consists of Est2p (catalytic subunit), the RNA component TLC1, and two accessory proteins, Est1p and Est3p (50). Cells deficient for any of these telomerase components undergo progressive telomere shortening and a simultaneous decrease in growth rate, described as senescence (24, 27). Typically, a small fraction of cells, termed survivors, escape senescence and maintain telomere length by utilizing RAD52-dependent recombination (24, 26).In addition to the telomerase complex, a number of yeast proteins are important in maintaining telomere length and integrity. These include Tel1p and Mec1p, the yeast homologues of mammalian ATM and ATR, respectively (39). While deletion of TEL1 results in short but stable telomeres, MEC1 deletion has little effect on average telomere length. However, cells lacking TEL1 that have a mutant mec1-21 allele undergo senescence, similar to telomerase null cells (36), suggesting that MEC1 plays a minor but essential role in telomere length maintenance in tel1Δ cells. It has been shown that the protein kinase activities of Tel1p and Mec1p are essential in telomere maintenance, since tel1^KD cells have short telomeres and tel1Δ mec1^KD cells undergo senescence (29).In current models, Tel1p acts to maintain telomere length by regulating the access of telomerase to short telomeres. TEL1 is required for the association of Est1p and Est2p with telomeres in the late S/G₂ phase of the cell cycle (16), the time when telomeres are elongated (9, 31). Additionally, in both yeast and mammalian cells, telomerase preferentially elongates the shortest telomeres (22, 30, 47). Therefore, TEL1 seems to be required mainly for the association of telomerase to short telomeres in yeast. Indeed, Tel1p preferentially binds to short telomeres (4, 21, 38) and is essential for the increased association of Est1p and Est2p to short telomeres during late S/G₂ (38). However, the kinase activity of Tel1p is not required for the telomere association (21). In addition to its role in telomerase recruitment, TEL1 may also regulate telomere length by enhancing the processivity of telomerase at short telomeres (7).The Mre11p, Rad50p, and Xrs2p (MRX) complex also plays important roles in telomere maintenance. Cells lacking any one of these components (mrxΔ) have short and stable telomeres. Since combining mrxΔ with tel1Δ has no synergistic effect on telomere shortening and mrxΔ mec1Δ cells undergo senescence, it was proposed that the MRX complex and Tel1p function in the same telomere maintenance pathway (37). In agreement with this model, the C-terminal region of Xrs2p is essential in recruiting Tel1p both to double-strand breaks (32) and to short telomeres (38). Interestingly, the mammalian functional homologue of Xrs2p, NBS1, interacts with ATM via its extreme C terminus (13), suggesting that the recruitment of Tel1p to telomeres and the recruitment of ATM to DNA damage sites are conserved.It remains a question what exact roles the kinase activity of Tel1p and its telomere binding play in telomere maintenance. Tel1p''s telomere maintenance function seems to be dependent on its kinase activity, since tel1^KD cells have short telomeres (29). It has been proposed that Tel1p may regulate the recruitment of Est1p, and thus the rest of the telomerase complex (12, 23, 54), to telomeres by phosphorylating Cdc13p (3, 48). Other experiments suggest the association of Tel1p to the telomere plays a major role. The preferential binding of Tel1p to short telomeres is lost in xrs2-664 cells (38), which lack the C-terminal 190 amino acids of Xrs2p and have short telomeres, similar to xrs2Δ (41). It has been suggested that the association of Tel1p to telomeres is required for its substrate phosphorylation and, therefore, telomere length maintenance (3, 39).To further analyze the functions of Tel1p in telomere maintenance, we generated a novel kinase-dead allele of TEL1 and new alleles of XRS2 that do not interact with Tel1p. Through these separation-of-function mutants, we show that both sets of alleles are partially active in a de novo telomere elongation assay. However, combining both the tel1^KD and either of the Tel1p interaction-deficient xrs2 alleles resulted in a phenotype resembling the tel1Δ phenotype, suggesting that Tel1p has kinase-dependent and kinase-independent, but telomere binding-dependent, functions in telomere maintenance.     

Rad50 Is Dispensable for the Maintenance and Viability of Postmitotic Tissues

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.The Mre11 complex regulates both DNA damage checkpoint function and repair. Its checkpoint functions appear to be primarily related to its role as a DNA double-strand break (DSB) sensor which binds DNA damage and activates ATM (ataxia-telangiectasia [AT] mutated). The ATM kinase transduces the damage signal via phosphorylating mediators of the damage response (30, 42), which promotes cell cycle arrest, DNA repair, and apoptosis. Mre11 complex functions are compromised in the human chromosome instability syndromes Nijmegen breakage syndrome and AT-like disorder, which are caused by hypomorphic mutations in Nbs1 and Mre11. Cells derived from patients and from mouse models of these diseases exhibit spontaneous DNA damage, ionizing radiation (IR) sensitivity, and checkpoint defects (25, 27, 48, 52, 57).The complex''s primary role in DNA repair is in recombinational DSB repair, and this role likely underlies its essential nature. In Saccharomyces cerevisiae, the complex governs homologous recombination (HR) and nonhomologous end joining (NHEJ) (19), whereas in vertebrate systems it primarily functions in HR (51, 61, 62). In fact, studies of Nbs1-deficient cells suggest that the Mre11 complex may inhibit NHEJ in mammals (62). Data from several species also implicate the Mre11 nuclease in the metabolism of topoisomerase adducts (40, 43, 49). This highly conserved function could also explain why the Mre11 complex is essential.The Mre11 complex''s function at telomeres may also be required for viability. Telomeres protect the ends of linear chromosomes from being recognized as DSBs and thereby activating the DNA damage response (DDR) (9). In S. cerevisiae the Mre11 complex influences telomere length maintenance (5, 28), whereas in mammals the complex interacts with the telomere binding protein Trf2 and localizes to telomeres (63). Loss of Trf2 results in telomere uncapping, causing activation of the DDR, telomere fusions, and senescence (7). Given the association of Mre11 with Trf2, it is conceivable that acute Mre11 complex deficiency in the mouse would phenocopy Trf2 loss and similarly lead to cell death as a result of telomere uncapping.Conclusions regarding the essential nature of HR in general (33, 47, 53) and the Mre11 complex specifically (10, 17, 45, 59, 62) have been derived from the analysis of proliferating cells in vitro or in vivo. The coincidence of DNA replication and the formation of spontaneous DSBs prompted us to test whether the Mre11 complex and, by extension, HR would be essential in quiescent or postmitotic tissues in which the frequency of spontaneous DSBs is significantly reduced. To examine this issue, we generated mice containing a conditional Rad50 allele in which the Rad50 gene could be inactivated in quiescent and postmitotic cells.Our results indicate that Rad50 is not required for homeostasis or viability of quiescent hepatocytes of the adult liver; nor does it appear to be required for maintenance of postmitotic Purkinje cells of the cerebellum. In contrast, Rad50 was required for viability of proliferating tissue culture and bone marrow cells. Rad50-deficient hepatocytes that were induced to divide via hepatectomy were able to achieve limited division and survived despite the presence of DNA damage that persisted long after the bulk of regeneration was complete. Rad50-deficient cells did not exhibit overtly dysfunctional telomeres, suggesting that their loss of viability was not due to acute telomere failure. These data indicate that the Mre11 complex and, by extension, HR may be dispensable in postmitotic cells and are consistent with the interpretation that the replication-associated functions of the Mre11 complex account for its essential nature.     

Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks

Rad3, the Schizosaccharomyces pombe ortholog of human ATR and Saccharomyces cerevisiae Mec1, activates the checkpoint kinase Chk1 in response to DNA double-strand breaks (DSBs). Rad3^ATR/Mec1 associates with replication protein A (RPA), which binds single-stranded DNA overhangs formed by DSB resection. In humans and both yeasts, DSBs are initially detected and processed by the Mre11-Rad50-Nbs1^Xrs2 (MRN) nucleolytic protein complex in association with the Tel1^ATM checkpoint kinase and the Ctp1^CtIP/Sae2 DNA-end processing factor; however, in budding yeast, neither Mre11 nuclease activity or Sae2 are required for Mec1 signaling at irreparable DSBs. Here, we investigate the relationship between DNA end processing and the DSB checkpoint response in fission yeast, and we report that Mre11 nuclease activity and Ctp1 are critical for efficient Rad3-to-Chk1 signaling. Moreover, deleting Ctp1 reveals a Tel1-to-Chk1 signaling pathway that bypasses Rad3. This pathway requires Mre11 nuclease activity, the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp complex, and Crb2 checkpoint mediator. Ctp1 negatively regulates this pathway by controlling MRN residency at DSBs. A Tel1-to-Chk1 checkpoint pathway acting at unresected DSBs provides a mechanism for coupling Chk1 activation to the initial detection of DSBs and suggests that ATM may activate Chk1 by both direct and indirect mechanisms in mammalian cells.DNA double-strand breaks (DSBs), formed by clastogens or from endogenous damage, trigger multiple cellular responses that are critical for maintaining genome integrity. Of particular importance is the cell cycle checkpoint that restrains the onset of mitosis while DSB repair is under way. Chk1 is the critical effector of this checkpoint in the fission yeast Schizosaccharomyces pombe and mammalian cells, whereas the budding yeast Saccharomyces cerevisiae uses both Chk1 and Rad53 (orthologous to human Chk2 and fission yeast Cds1) to delay anaphase entry and mitotic exit. These kinases are regulated by ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) checkpoint kinases (5). Curiously, the regulatory connections between ATM/ATR and Chk1/Chk2 orthologs are not strictly conserved between species (Fig. ​(Fig.1A).1A). In mammals, ATM activates Chk2 while ATR activates Chk1. In S. cerevisiae and S. pombe, ATR orthologs (Mec1 and Rad3, respectively) activate Chk2 orthologs and Chk1, while Tel1 (ATM ortholog) is primarily involved in telomere maintenance (14, 38, 40).Open in a separate windowFIG. 1.Deletion of Ctp1 restores the DNA damage checkpoint in rad3Δ cells. (A) Regulatory connections between ATM/ATR and Chk1/Chk2 orthologs in mammals, S. cerevisiae, and S. pombe. ATM phosphorylates Chk2 and ATR phosphorylates Chk1. CtIP mediates an ATM-to-ATR switch through DNA end resection in mammals (44, 53). ATM promotes Chk1 activation by stimulating CtIP-dependent resection through an unknown mechanism. In S. cerevisiae, Mec1 phosphorylates both Rad53 and Chk1. Deleting Sae2 uncovers a Tel1-to-Rad53 signaling pathway and enhances Rad53 activation (47). In S. pombe, Cds1 and Chk1 activation is dependent on Rad3. (B) Chk1 phosphorylation peaks in wild-type (wt) (top panel) and ctp1Δ cells (bottom panel) 30 min after exposure to 90 Gy of IR in log-phase cultures. Chk1 phosphorylation in ctp1Δ cells prior to IR exposure likely arises from an inability to repair spontaneous DNA damage (23). Immunoblots were probed for the HA epitope-tagged Chk1 or Cdc2 as a loading control. (C) Chk1 phosphorylation is reduced at least 2-fold in ctp1Δ cells relative to the wild type. Quantification of blots from panel B expressed as a ratio of phospho-Chk1 (upper band) versus nonphospho-Chk1 (lower band) was performed. The phospho-Chk1 signal in untreated ctp1Δ cells was subtracted from the IR-treated samples to more accurately measure the IR-induced phosphorylation. (D) The ctp1Δ mutation restores Chk1 phosphorylation in rad3Δ cells. Cells were harvested immediately after mock or 90-Gy IR treatment and blotted for HA epitope tag. Ponceau staining shows equal loading. (E) Quantitation of Chk1 phosphorylation. Error bars represent the standard errors from three independent experiments. (F) The checkpoint arrest is restored in ctp1Δ rad3Δ cells. Cells synchronized in G₂ by elutriation were mock treated or exposed to 100 Gy of IR. Cell cycle progression was tracked by microscopic observation.The functions of ATM and ATR orthologs are intimately tied to the detection and nucleolytic processing of DSBs. ATM^Tel1 localizes at DSBs by interacting with Mre11-Rad50-Nbs1^Xrs2 (MRN) protein complex, which directly binds DNA ends (12, 20, 24, 50, 52). The MRN complex is essential for ATM^Tel1 function in all species. The Mre11 subunit of MRN complex has DNase activities that are critical for radioresistance in S. pombe and mice but not in budding yeast (3, 19, 22, 50). In fission yeast, MRN complex also recruits Ctp1 DNA end-processing factor to DSBs (25, 49). Ctp1 is structurally and functionally related to CtIP in mammals and Sae2 in budding yeast, the latter of which has nuclease activity in vitro (21, 23, 43). Ctp1 and CtIP are essential for survival of ionizing radiation and other clastogens (23, 43, 54), whereas sae2Δ mutants are not radiosensitive except at very high doses of ionizing radiation (IR), although both Ctp1 and Sae2 are required for repair of meiotic DSBs formed by a Spo11/Rec12-dependent mechanism (17, 23, 36). Genetic and biochemical studies indicate that Sae2/Ctp1/CtIP collaborate with MRN complex to initiate the 5′-to-3′ resection of DSBs (7, 23, 28, 43, 53, 55), which leads to the generation of 3′ single-strand overhangs (SSOs) that are critical for DSB repair by homologous recombination (HR). Replication protein A (RPA) binding to SSOs is essential for HR repair of DSBs, but it is also important for recruiting ATR^Rad3/Mec1, which interacts with RPA through its regulatory subunit ATRIP (Rad26 in fission yeast, Ddc2 in budding yeast) (5, 56). Subsequent phosphorylation of Chk1 by ATR also requires the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp, which is loaded at the single-strand/double-strand DNA junctions (26, 48, 57), the ATR activating protein TopBP1 (Cut5 in fission yeast), and a checkpoint mediator protein such as Crb2 in fission yeast (34, 41, 48).In this mechanism of DNA damage checkpoint signaling, DNA end resection is critical for ATR (Rad3/Mec1) activation, and therefore resection defective mutants should be unable to mount a fully active checkpoint response (44). However, Rad53 activation is not diminished in budding yeast sae2Δ mutants that suffer an irreparable DSB by expressing HO endonuclease. In fact, there is a defect in turning off the checkpoint signal (6). A similar effect is observed in S. cerevisiae strains expressing the mre11-H125N nuclease-defective form of Mre11. Moreover, overexpression of SAE2 strongly inhibits Rad53 activation (6). The reasons for these phenotypes are unknown, since neither Sae2 nor Mre11 nuclease activity are required for DSB resection or radioresistance. However, deleting Sae2 delays resection while at the same time enhancing a cryptic Tel1-to-Rad53 checkpoint pathway (6, 47). These effects correlate with delayed disassembly of Mre11 foci at DSBs in sae2Δ cells, suggesting that Sae2 may negatively regulate checkpoint signaling by modulating Mre11 association at damaged DNA (1, 6, 24). Enhancement of a Tel1-to-Rad53 checkpoint pathway by eliminating Sae2 suggests that the signaling pathways between ATM/ATR and Chk1/Chk2 checkpoint kinases are not hard wired but are adaptable to changes in DNA end processing (47). However, as yet there is no evidence that ATM^Tel1 can activate Chk1 in any organism.Since SAE2 deletion or overexpression has unexpected effects on Rad53 activation in budding yeast, we decided to explore the relationship between Ctp1 and Chk1 activation in fission yeast. Here, we show that Chk1 activation is substantially diminished in ctp1Δ cells exposed to ionizing radiation. These data are consistent with studies showing that CtIP is required for efficient Chk1 activation in mammalian cells treated with camptothecin (CPT), a topoisomerase I poison that causes replication fork collapse (43, 53). We also investigate the role of Mre11 nuclease activity and find that while ablating Mre11 nuclease activity enhances Rad53 activation in budding yeast, the equivalent Mre11 mutation in fission yeast severely impairs Chk1 activation by ionizing radiation. Furthermore, we find that deleting Ctp1 reveals a previously unknown Tel1-to-Chk1 signaling pathway in S. pombe, a finding analogous to the enhancement of a Tel1-to-Rad53 checkpoint pathway by eliminating Sae2 in S. cerevisiae (47). This Tel1-to-Chk1 pathway also requires Mre11 nuclease activity. These data establish that Tel1^ATM can activate Chk1 independently of Rad3^ATR, which has implications for studies linking ATM to Chk1 activation in mammalian cells (16, 31). Characterization of this pathway allows us to propose a more detailed model of how Chk1 is activated in response to DSBs.     

Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G1 and Resection-Mediated Inhibition of NHEJ in G2

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G₁, consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G₂, specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3′ overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G₂ through end resection and induction of NHEJ in G₁ through ATM-dependent signaling.Mammalian telomeres solve the end protection problem through their association with shelterin. The shelterin factor TRF2 (telomere repeat-binding factor 2) protects chromosome ends from inappropriate DNA repair events that threaten the integrity of the genome (reviewed in reference 32). When TRF2 is removed by Cre-mediated deletion from conditional knockout mouse embryo fibroblasts (TRF2^F/− MEFs), telomeres activate the ATM kinase pathway and are processed by the canonical nonhomologous end-joining (NHEJ) pathway to generate chromosome end-to-end fusions (10, 11).The repair of telomeres in TRF2-deficient cells is readily monitored in metaphase spreads. Over the course of four or five cell divisions, the majority of chromosome ends become fused, resulting in metaphase spreads displaying the typical pattern of long trains of joined chromosomes (10). The reproducible pace and the efficiency of telomere NHEJ have allowed the study of factors involved in its execution and regulation. In addition to depending on the NHEJ factors Ku70 and DNA ligase IV (10, 11), telomere fusions are facilitated by the ATM kinase (26). This aspect of telomere NHEJ is mediated through the ATM kinase target 53BP1. 53BP1 accumulates at telomeres in TRF2-depleted cells and stimulates chromatin mobility, thereby promoting the juxtaposition of distantly positioned chromosome ends prior to their fusion (18). Telomere NHEJ is also accelerated by the ATM phosphorylation target MDC1, which is required for the prolonged association of 53BP1 at sites of DNA damage (19).Although loss of TRF2 leads to telomere deprotection at all stages of the cell cycle, NHEJ of uncapped telomeres takes place primarily before their replication in G₁ (25). Postreplicative (G₂) telomere fusions can occur at a low frequency upon TRF2 deletion, but only when cyclin-dependent kinase activity is inhibited with roscovitine (25). The target of Cdk1 in this setting is not known.Here, we dissect the role of the MRN (Mre11/Rad50/Nbs1) complex and H2AX at telomeres rendered dysfunctional through deletion of TRF2. The highly conserved MRN complex has been proposed to function as the double-stranded break (DSB) sensor in the ATM pathway (reviewed in references 34 and 35). In support of this model, Mre11 interacts directly with DNA ends via two carboxy-terminal DNA binding domains (13, 14); the recruitment of MRN to sites of damage is independent of ATM signaling, as it occurs in the presence of the phosphoinositide-3-kinase-related protein kinase inhibitor caffeine (29, 44); in vitro analysis has demonstrated that MRN is required for activation of ATM by linear DNAs (27); a mutant form of Rad50 (Rad50S) can induce ATM signaling in the absence of DNA damage (31); and phosphorylation of ATM targets in response to ionizing radiation is completely abrogated upon deletion of NBS1 from MEFs (17). These data and the striking similarities between syndromes caused by mutations in ATM, Nbs1, and Mre11 (ataxia telangiectasia, Nijmegen breakage syndrome, and ataxia telangiectasia-like disease, respectively) are consistent with a sensor function for MRN.MRN has also been implicated in several aspects of DNA repair. Potentially relevant to DNA repair events, Mre11 dimers can bridge and align the two DNA ends in vitro (49) and Rad50 may promote long-range tethering of sister chromatids (24, 50). In addition, a binding partner of the MRN complex, CtIP, has been implicated in end resection of DNA ends during homology-directed repair (39, 45). The role of MRN in NHEJ has been much less clear. MRX, the yeast orthologue of MRN, functions during NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe (28, 30). In mammalian cells, MRN is not recruited to I-SceI-induced DSBs in G₁, whereas Ku70 is, and MRN does not appear to be required for NHEJ-mediated repair of these DSBs (38, 54). On the other hand, MRN promotes class switch recombination (37) and has been implicated in accurate NHEJ repair during V(D)J recombination (22).The involvement of MRN in ATM signaling and DNA repair pathways has been intriguing from the perspective of telomere biology. While several of the attributes of MRN might be considered a threat to telomere integrity, MRN is known to associate with mammalian telomeres, most likely through an interaction with the TRF2 complex (48, 51, 57). MRN has been implicated in the generation of the telomeric overhang (12), the telomerase pathway (36, 52), the ALT pathway (55), and the protection of telomeres from stochastic deletion events (1). It has also been speculated that MRN may contribute to formation of the t-loop structure (16). t-loops, the lariats formed through the strand invasion of the telomere terminus into the duplex telomeric DNA (21), are thought to contribute to telomere protection by effectively shielding the chromosome end from DNA damage response factors that interact with DNA ends, including nucleases, and the Ku heterodimer (15).H2AX has been studied extensively in the context of chromosome-internal DSBs. When a DSB is formed, ATM acts near the lesion to phosphorylate a conserved carboxy-terminal serine of H2AX, a histone variant present throughout the genome (7). Phosphorylated H2AX (referred to as γ-H2AX) promotes the spreading of DNA damage factors over several megabases along the damaged chromatin and mediates the amplification of the DNA damage signal (43). The signal amplification is accomplished through a sequence of phospho-specific interactions among γ-H2AX, MDC1, NBS1, RNF8, and RNF168, which results in the additional binding of ATM and additional phosphorylation of H2AX in adjacent chromatin (reviewed in reference 33). The formation of these large domains of altered chromatin, referred to as irradiation-induced foci at DSBs and telomere dysfunction-induced foci (TIFs) at dysfunctional telomeres (44), promotes the binding of several factors implicated in DNA repair, including the BRCA1 A complex and 53BP1 (33).In agreement with a role for H2AX in DNA repair, H2AX-deficient cells exhibit elevated levels of irradiation-induced chromosome abnormalities (5, 9). In addition, H2AX-null B cells are prone to chromosome breaks and translocations in the immunoglobulin locus, indicative of impaired class switch recombination, a process that involves the repair of DSBs through the NHEJ pathway (9, 20). Since H2AX is dispensable for the activation of irradiation-induced checkpoints (8), these data argue that H2AX contributes directly to DNA repair. However, a different set of studies has concluded that H2AX is not required for NHEJ during V(D)J recombination (5, 9) but that it plays a role in homology-directed repair (53). In this study, we have further queried the contribution of H2AX to NHEJ in the context of dysfunctional telomeres.Our aim was to dissect the contribution of MRN and H2AX to DNA damage signaling and NHEJ-mediated repair in response to telomere dysfunction elicited by deletion of TRF2. Importantly, since ATM is the only kinase activated in this setting, deletion of TRF2 can illuminate the specific contribution of these factors in the absence of the confounding effects of ATR signaling (26). This approach revealed a dual role for MRN at telomeres, involving both its function as a sensor in the ATM pathway and its ability to protect telomeres from NHEJ under certain circumstances.     

Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.Yeast sporulation is a developmental process that involves multiple, sequential events that need to be tightly coordinated (59, 68). In the fission yeast Schizosaccharomyces pombe, when cells of opposite mating type (h⁺ and h⁻) are mixed and shifted to conditions of nitrogen starvation, cell fusion and karyogamy occur to form a diploid zygote, which then undergoes premeiotic DNA replication, the two meiotic divisions, formation of the spore envelopes (comprising the plasma membrane and a specialized cell wall), and maturation of the spores (74, 81). At the onset of meiosis II, precursors of the spore envelopes, the forespore membranes (FSMs), are formed by the fusion of vesicles at the cytoplasmic surface of each spindle-pole body (SPB) and then extend to engulf the four nuclear lobes (the nuclear envelope does not break down during meiosis), thus capturing the haploid nuclei, along with associated cytoplasm and organelles, to form the nascent spores (55, 68, 81). How the FSMs recognize and interact with the nuclear envelope, extend in a properly oriented manner, and close to form uniformly sized spherical spores is not understood, and study of this model system should also help to elucidate the more general question of how membranes obtain their shapes in vivo.It has been shown that both the SPB and the vesicle trafficking system play important roles in the formation and development of the FSM and of its counterpart in the budding yeast Saccharomyces cerevisiae, the prospore membrane (PSM). In S. pombe, the SPB changes its shape from a compact dot to a crescent at metaphase of meiosis II (26, 29), and its outer plaque acquires meiosis-specific components such as Spo2, Spo13, and Spo15 (30, 57, 68). This modified outer plaque is required for the initiation of FSM assembly. In S. cerevisiae, it is well established that various secretory (SEC) gene products are required for PSM formation (58, 59). Similarly, proteins presumably involved in the docking and/or fusion of post-Golgi vesicles and organelles in S. pombe, such as the syntaxin-1A Psy1, the SNAP-25 homologue Sec9, and the Rab7 GTPase homologue Ypt7, are also required for proper FSM extension (34, 53, 54). Consistent with this hypothesis, Psy1 disappears from the plasma membrane upon exit from meiosis I and reappears in the nascent FSM.Phosphoinositide-mediated membrane trafficking also contributes to the development of the FSM. Pik3/Vps34 is a phosphatidylinositol 3-kinase whose product is phosphatidylinositol 3-phosphate [PtdIns(3)P] (35, 72). S. pombe cells lacking this protein exhibit defects in various steps of FSM formation, such as aberrant starting positions for extension, disoriented extension and/or failure of closure, and the formation of spore-like bodies near, rather than surrounding, the nuclei, suggesting that Pik3 plays multiple roles during sporulation (61). The targets of PtdIns(3)P during sporulation appear to include two sorting nexins, Vps5 and Vps17, and the FYVE domain-containing protein Sst4/Vps27. vps5Δ and vps17Δ mutant cells share some of the phenotypes of pik3Δ cells (38). sst4Δ cells also share some of the phenotypes of pik3Δ cells but are distinct from vps5Δ and vps17Δ cells, consistent with the hypothesis that Pik3 has multiple roles during sporulation (62).Membrane trafficking processes alone do not seem sufficient to explain how the FSMs and PSMs extend around and engulf the nuclei, suggesting that some other mechanism(s) must regulate and orient FSM/PSM extension. The observation that the FSM is attached to the SPB until formation of the immature spore is complete (68) suggests that the SPB may regulate FSM extension. In addition, the leading edge of the S. cerevisiae PSM is coated with a complex of proteins (the LEPs) that appear to be involved in PSM extension (51, 59). S. pombe Meu14 also localizes to the leading edge of the FSM, and deletion of meu14 causes aberrant FSM formation in addition to a failure in SPB modification (60). However, it has remained unclear whether the SPB- and LEP-based mechanisms are sufficient to account for the formation of closed FSMs and PSMs of proper size and position (relative to the nuclear envelope), and evidence from S. cerevisiae has suggested that the septin proteins may also be involved.The septins are a conserved family of GTP-binding proteins that were first identified in S. cerevisiae by analysis of the cytokinesis-defective cdc3, cdc10, cdc11, and cdc12 mutants (41). Cdc3, Cdc10, Cdc11, and Cdc12 are related to each other in sequence and form an oligomeric complex that localizes to a ring in close apposition to the plasma membrane at the mother-bud neck in vegetative cells (12, 20, 25, 41, 47, 77). The septin ring appears to be filamentous in vivo (12), and indeed, the septins from both yeast (11, 20) and metazoans (31, 36, 69) can form filaments in vitro. The yeast septin ring appears to form a scaffold for the localization and organization of a wide variety of other proteins (8, 22), and it forms a diffusion barrier that constrains movement of membrane proteins through the neck region (7, 8, 73). In metazoan cells, the septins are involved in cytokinesis but are also implicated in a variety of other cellular processes, such as vesicular transport, organization of the actin and microtubule cytoskeletons, and oncogenesis (27, 70).In S. cerevisiae, a fifth septin (Shs1) is also expressed in vegetative cells, but the remaining two septin genes, SPR3 and SPR28, are expressed at detectable levels only during sporulation (15, 17). In addition, at least some of the vegetatively expressed septins are also present in sporulating cells (17, 48), and one of them (Cdc10) is expressed at much higher levels there than in vegetative cells (32). The septins present during sporulation are associated with the PSM (15, 17, 48, 51), and their normal organization there depends on the Gip1-Glc7 protein phosphatase complex (71). However, it has been difficult to gain insight into the precise roles of the septins during sporulation in S. cerevisiae (59), because some septins are essential for viability during vegetative growth, and the viable mutants have only mild phenotypes during sporulation (15, 17), possibly because of functional redundancy among the multiple septins.S. pombe seemed likely to provide a better opportunity for investigating the role of septins during spore formation. There are seven septin genes (spn1⁺ to spn7⁺) in this organism (23, 41, 63). Four of these genes (spn1⁺ to spn4⁺) are expressed in vegetative cells, and their products form a hetero-oligomeric complex that assembles during cytokinesis into a ring at the division site (2, 3, 10, 76, 79). The septin ring is important for proper targeting of endoglucanases to the division site (44), and septin mutants show a corresponding delay in cell separation (10, 41, 44, 76). However, even the spn1Δ spn2Δ spn3Δ spn4Δ quadruple mutant is viable and grows nearly as rapidly as the wild type (our unpublished results), a circumstance that greatly facilitates studies of the septins'' role during sporulation.spn5⁺, spn6⁺, and spn7⁺ are expressed at detectable levels only during sporulation (1, 45, 78; our unpublished results), and spn2⁺, like its orthologue CDC10 (see above), is strongly induced (45), but the roles of the S. pombe septins in sporulation have not previously been investigated. In this study, we show that the septins are important for the orientation of FSM extension, suggesting that the septins may have a more general role in dynamic membrane organization and shape determination.     

Structural and Functional Characterization of Diffusible Signal Factor Family Quorum-Sensing Signals Produced by Members of the Burkholderia cepacia Complex

Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis-2-dodecenoic acid (C₁₂:Δ², also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C₁₂:Δ² production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C₁₂:Δ² as a sole DSF family signal, while four species produce not only C₁₂:Δ² but also a new DSF family signal, which was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5). In addition, we demonstrate that the quorum-sensing signal cis-11-methyl-2-dodecenoic acid (11-Me-C₁₂:Δ²), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans. It is shown that, similar to 11-Me-C₁₂:Δ² and C₁₂:Δ², the newly identified molecule 11-Me-C₁₂:Δ^2,5 is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans. These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.The Burkholderia cepacia complex (Bcc) comprises a group of currently 17 formally named bacterial species that, although closely related, are phenotypically diverse (17, 22, 23). Strains of the Bcc are ubiquitously distributed in nature and have been isolated from soil, water, the rhizosphere of plants, industrial settings, hospital environments, and infected humans. Some Bcc strains have emerged as problematic opportunistic pathogens in patients with cystic fibrosis or chronic granulomatous disease, as well as in immunocompromised individuals (17). The clinical outcome of Bcc infections ranges from asymptomatic carriage to a fulminant and fatal pneumonia, the so-called “cepacia syndrome” (12, 17). Although all Bcc species have been isolated from both environmental and clinical sources, B. cenocepacia and B. multivorans are most commonly found in clinical samples (16).Many bacterial pathogens have evolved a cell-cell communication mechanism known as quorum sensing (QS) to coordinate the expression of virulence genes. In spite of their genetic differences, most Bcc species produce N-acylhomoserine lactone (AHL) QS signals (25). More recently, another QS signal molecule, cis-2-dodecenoic acid (BDSF), has been identified in B. cenocepacia (3). Subsequent studies showed that BDSF plays a role in the regulation of bacterial virulence (6, 20). Interestingly, the two QS systems appear to act in conjunction in the regulation of B. cenocepacia virulence, as a set of the AHL-controlled virulence genes are also positively regulated by BDSF (6). Furthermore, mutation of Bcam0581, which is required for BDSF biosynthesis, results in substantially retarded energy production and impaired growth in minimal medium (6), highlighting the dual roles of the QS system in the physiology of and infection by B. cenocepacia.BDSF is a structural analogue of cis-11-methyl-2-dodecenoic acid, which is a QS signal known as diffusible signal factor (DSF) originally identified from the plant bacterial pathogen Xanthomonas campestris pv. campestris (2, 24). Evidence is accumulating that DSF-type fatty acid signals represent a new family of QS signals, which are widespread among Gram-negative bacteria (10, 24). For example, DSF and seven structural derivatives were identified in supernatants of Stenotrophomonas maltophilia (8, 11), 12-methyl-tetradecanoic acid was shown to be produced by Xylella fastidiosa (18), and cis-2-decenocic acid was found to be synthesized by Pseudomonas aeruginosa (5). In addition, DSF-like activity has also been reported in a range of Xanthomonas species, including X. oryzae pv. oryzae and X. axonopodis pv. citri (1, 2, 4, 24), but the chemical structures of these DSF analogues remain to be determined. Unlike other known QS signals, such as AHL and AI-2 family signals, DSF and its analogues, including BDSF, are fatty acids and these fatty acid signals were collectively designated DSF family signals for the convenience of discussion (10). Considering the fact that the list of DSF family signal is expanding, we propose to designate cis-11-methyl-2-dodecenoic acid (DSF) 11-Me-C₁₂:Δ² and cis-2-dodecenoic acid (BDSF) C₁₂:Δ². This nomenclature is based on one of the fatty acid nomenclatures (13, 19) where the methyl (Me) substitution and its position are indicated first (for example, 11-Me indicates a methyl group on C-11 of the fatty acid carbon chain), followed by the length of the fatty acid carbon chain (C₁₂ represents a 12-carbon fatty acid chain), and then the position of the double bond in the fatty acid chain (Δ² indicates a double bond in the cis configuration at site 2, i.e., between C-2 and C-3 of the fatty acid carbon chain). In this way, it is convenient to say that 11-Me-C₁₂:Δ² and C₁₂:Δ² have identical 12-carbon fatty acid chains with a cis bond at the same site but differ in a methyl substitution on C-11. Following this nomenclature system, 12-methyl-tetradecanoic acid and cis-2-decenocic acid can be referred to as 12-Me-C₁₄ and C₁₀:Δ², respectively.DSF family signals have emerged as important factors in the regulation of virulence and biofilm formation in a wide range of bacterial pathogens (10). In this study, we have investigated the production of the DSF family signals in nine Bcc species. It is demonstrated that C₁₂:Δ² is conserved in members of the Bcc and that 11-Me-C₁₂:Δ² and a novel DSF family signal were also produced by some, but not all, of the Bcc strains investigated. This new signal was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5) by nuclear magnetic resonance (NMR) analysis and mass spectrometry. We have also investigated the biological significance of 11-Me-C₁₂:Δ^2,5 in intraspecies and interspecies communication.     

BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3^ATR, forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT₂) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1^ATM and Rad3^ATR, although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT₂ domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT₂ γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.Double-strand breaks (DSBs) are among the most dangerous forms of DNA damage (26, 30). Human cells experience DSBs several times a day, either during normal metabolism or as a consequence of exposure to DNA-damaging agents, such as ionizing radiation (IR) (18). Importantly, the unfaithful repair of such breaks can result in genome instability and cancer. The response to DSBs is coordinated by a conserved signal transduction cascade, which leads to cell cycle arrest and activation of DNA repair and constitutes the checkpoint response (9, 14, 20). The essential players in this process fall into four groups: sensors, mediators, transducers, and effectors (20). Sensors are the first to recognize and bind to DNA breaks and include the Mre11-Rad50-Nbs1 complex in humans and Schizosaccharomyces pombe (Mre11-Rad50-Xrs2 in Saccharomyces cerevisiae). The PIKKs (phosphoinositide 3-kinase-like kinases) ATR-ATRIP (ScMec1-ScDdc2/SpRad3-SpRad26) and ATM (ScTel1/SpTel1) act as transducers that transmit the signal to the effector kinases Chk1 (ScChk1/SpChk1) and Chk2 (ScRad53/SpCds1), whose role is to target downstream targets, such as p53 in mammals, and to amplify the signal (9, 14, 20).Signaling between transducers and effectors is facilitated and enhanced by mediator proteins (19, 20). In the fission yeast Schizosaccharomyces pombe, Crb2/Rhp9 is a critical mediator of the DNA damage checkpoint (31, 42) and is related to Saccharomyces cerevisiae Rad9 and mammalian 53BP1 (p53 binding protein 1). Rad3^ATR-Rad26^ATRIP phosphorylates Crb2 in response to damage, and Crb2 is required for phosphorylation of Chk1 by Rad3^ATR-Rad26^ATRIP (31). Chk1, in turn, restrains entry into mitosis by phosphorylating and thus inactivating the phosphatase Cdc25 that is a mitotic inducer (10, 11, 28). Crb2-null cells are sensitive to a range of genotoxins and are unable to delay division in response to DNA damage (31, 42).Crb2 is a nuclear protein that rapidly relocalizes to DSBs. This occurs on such a large scale that IR-induced nuclear foci (IRIF) of yellow fluorescent protein (YFP)-tagged Crb2 expressed from the endogenous promoter are readily detected by live cell microscopy (5). These foci colocalize with homologous recombination (HR) repair factors such as Rad22^Rad52. Two types of histone modifications regulate Crb2 localization at DSBs: C-terminal phosphorylation of histone H2A, denoted as γ-H2A (23), and lysine-20 dimethylation of histone H4, denoted as H4-K20me2 (32). Phosphorylation of an SQ motif within the C-terminal tail of histone H2A of budding yeast or fission yeast, or the H2AX variant in mammals, is one of the earliest cellular responses triggered by DNA damage (3, 23, 29). The γ-H2A/X modification, which is catalyzed by the checkpoint kinases ATR^Rad3 and ATM^Tel1, spans large distances on both sides of a DSB, and it plays a critical role in recruiting DNA damage response proteins, chromatin remodeling complexes, and cohesin (2, 21, 23, 34, 35, 37, 38, 40). Protein crystallography and biochemical studies established that mammalian Mdc1, S. pombe Crb2, and Brc1 DNA damage response proteins directly bind the phosphorylated tail of histone H2A/X through tandem C-terminal BRCT domains (16, 35, 40). In contrast to γ-H2A, H4-K20 methylation catalyzed by Set9/Kmt5 histone methyltransferase appears to be constitutive and not regulated by DNA damage (32). H4-K20me2 directly binds tandem tudor domains (Tudor₂) located to the N-terminal side of the BRCT domains in Crb2 (1).YFP-Crb2 does not form IRIF in hta1-S129A hta2-S128A (htaAQ) or rad3Δ tel1Δ cells, in which γ-H2A phosphorylation is abolished (23), or in set9Δ cells or tudor domain mutants of Crb2 that ablate binding to H4-K20me2 (6, 32). However, Crb2 checkpoint functions are only partially impaired in an htaAQ set9Δ strain, implying that physiologically significant recruitment of Crb2 to DSBs also occurs by a histone modification-independent pathway. Indeed, we found that YFP-Crb2 forms microscopically visible foci in htaAQ set9Δ cells when DSBs are created by HO endonuclease or by treating cells in G₁ phase with IR (6). Unlike IR-induced DSBs formed during G₂ phase, these types of DSBs lack an intact sister chromatid that can be used for HR repair and therefore they are highly persistent. Further analysis revealed that the histone modification-independent pathway of recruiting Crb2 to DSBs requires threonine-215 (Thr215) phosphorylation catalyzed by the cyclin-dependent kinase (CDK) Cdc2, which facilitates an interaction with Cut5 (ScDpb11; mammalian TopBP1) (6, 8, 31). The crb2-T215A mutation does not ablate YFP-Crb2 IRIF formation; however, Crb2 Thr215 phosphorylation is required for formation of YFP-Crb2 foci at persistent DSBs in htaAQ or set9Δ cells, and combining crb2-T215A with htaAQ or set9Δ abolishes Crb2 function (6).The tandem C-terminal BRCT domains (BRCT₂) of Crb2 not only mediate interactions with γ-H2A but also coordinate Crb2 homodimerization (4). In fact, replacing BRCT₂ with a leucine zipper (LZ) dimerization motif restores substantial function to Crb2 without restoring its ability to form IRIF. Thus, the most crucial task of the Crb2 BRCT domains is to provide a homodimerization platform, while binding to γ-H2A provides an additional function that is necessary for full resistance to DNA damage (4).In a recent study, Kilkenny et al. (16) solved the crystal structures of Crb2-BRCT₂ alone and in complex with a γ-H2A-derived phosphopeptide containing the common C-terminal residues of H2A.1 and H2A.2 (the two H2A paralogues in S. pombe). These analyses revealed the structural determinants of BRCT₂ binding to γ-H2A and BRCT₂-mediated homodimerization of Crb2. Ser666 was found to be critical for homodimerization in vitro, and mutation of this residue severely impaired Crb2 function in vivo. Residues Ser548 and Lys619 were identified as important for the interaction with the phosphate group on γ-H2A.1 pSer129. However, a charge reversal mutation of Lys619 did not abrogate Crb2 IRIF formation measured using methanol-fixed cells, although it did disrupt binding to a γ-H2A peptide in vitro (16). These unexpected findings indicated that γ-H2A likely has an indirect role in regulating Crb2 localization at DSBs. Here, we investigate Crb2 localization in live cells and find that while mutations of Ser548 or Lys619 partially impair Crb2 IRIF, the corresponding double mutant is severely deficient in Crb2 IRIF formation. Our findings and an independent study by Sanders et al. (33) show that γ-H2A binding to BRCT₂ is critical for Crb2 focus formation at IR-induced DSBs and for maintaining a DNA damage checkpoint response.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.DNA damage arises from errors in the replication process, as well as a myriad of intrinsic and extrinsic DNA-damaging agents that continually assault cells. Failure to efficiently repair DNA lesions leads to accumulation of mutations that contribute to numerous pathologies, including carcinogenesis. In addition to genomic DNA, mitochondrial DNA (mtDNA) is subject to damage that requires repair to maintain integrity. For these reasons, it is not surprising that DNA replication and repair proteins display significant plasticity that allows participation in several divergent replication and repair processes. In addition, numerous mechanisms, including alternative splicing, posttranslational modifications, or utilization of alternative translation initiation start sites, allow DNA replication and repair proteins such as Pif1, DNA ligase III, and APE1 to localize to the nucleus and the mitochondrion and participate in DNA replication and/or repair (9, 17, 25), thus ensuring genomic DNA and mtDNA integrity.Dna2 is an evolutionarily conserved helicase/nuclease enzyme. Originally discovered in Saccharomyces cerevisiae, Dna2 orthologs are found throughout the animal kingdom, including humans (5, 22, 28). Early studies demonstrated that Dna2 functions in concert with Flap endonuclease 1 (FEN1) to remove long DNA flaps that form upon lagging-strand DNA replication (6). However, in contrast to FEN1, Dna2 is an essential gene in yeast, suggesting that other proteins, including FEN1, cannot compensate for its loss in DNA replication or that it possesses functions beyond its role in Okazaki fragment processing. In agreement with this, genetic and biochemical studies have implicated Dna2 in DNA double-strand break (DSB) repair, telomere regulation, and mitochondrial function (8, 10, 15, 26, 38, 44, 45).Analysis of Dna2 in yeast revealed that it undergoes dynamic cell cycle localization. Dna2 localizes to telomeres during G₁, relocalizes throughout the genome in S phase, and moves back to the telomere during late S/G₂, where it participates in telomere replication and telomerase-dependent telomere elongation (10). Dna2 also leaves the telomere following treatment with bleomycin and localizes to sites of DNA DSBs (10). In addition, dna2 mutants are sensitive to DNA damage induced by gamma radiation and methanesulfonic acid methyl ester (7, 15). These phenotypes may be explained by recent work demonstrating that Dna2 plays an important role in 5′-end resection following DSBs. Indeed, upon induction of DSBs and initiation of 5′-end resection by the Mre11-Rad50-Xrs2 complex, Dna2 and Sgs1 cooperate to further degrade the 5′ end, creating long 3′ strands essential for homologous recombination (26, 45). Finally, while dna2Δ mutations are lethal in budding yeast, the dna2Δ pif1-m2 (nuclear PIF1) double mutations rescue dna2Δ lethality but produce a petite phenotype, suggesting that Dna2 is also involved in mtDNA maintenance (8).Recently, the human ortholog of Dna2 was cloned and characterized (23, 29). Biochemical analysis revealed that, similar to its yeast counterpart, the human Dna2 (hDna2) protein possesses nuclease, ATPase, and limited helicase activities (23, 29), suggesting that it carries out analogous functions in yeast and mammalian cells. However, hDna2''s putative role in genomic DNA repair and replication was called into question by a recent study suggesting that hDna2 is absent from the nucleus and found exclusively within the mitochondria, where it participates in mtDNA repair (44). Further in vitro biochemical studies suggested that hDna2 also participates in mtDNA replication (44). Here, we confirm that hDna2 localizes to the mitochondria and demonstrate that hDna2 participates in mtDNA replication and repair. However, our studies go further by uncovering a nuclear form of hDna2 that plays an important role in genomic stability. Indeed, we demonstrate that depletion of hDna2 leads to the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that hDna2, like its yeast counterpart, is essential to maintain nuclear DNA stability.