DNA damage checkpoint recovery and cancer development.

Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients׳ poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression.

SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated repair.

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.

Cdc5L interacts with ATR and is required for the S-phase cell-cycle checkpoint.

Cell division cycle 5-like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre-messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell-cycle checkpoint kinase ataxia-telangiectasia and Rad3-related (ATR). Depletion of Cdc5L by RNA-mediated interference methods results in a defective S-phase cell-cycle checkpoint and cellular sensitivity in response to replication-fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR-binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L-depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR-mediated cell-cycle checkpoint in response to genotoxic agents.

Cyclin E is stabilized in response to replication fork barriers leading to prolonged S phase arrest.

Cyclin E is a regulator of cyclin-dependent protein kinases (Cdks) and is involved in mediating the cell cycle transition from G(1) to S phase. Here, we describe a novel function for cyclin E in the long term maintenance of checkpoint arrest in response to replication barriers. Exposure of cells to mitomycin C or UV irradiation, but not ionizing radiation, induces stabilization of cyclin E. Stabilization of cyclin E reduces the activity of Cdk2-cyclin A, resulting in a slowing of S phase progression and arrest. In addition, cyclin E is shown to be required for stabilization of Cdc6, which is required for activation of Chk1 and the replication checkpoint pathway. Furthermore, the stabilization of cyclin E in response to replication fork barriers depends on ATR, but not Nbs1 or Chk1. These results indicate that in addition to its well studied role in promoting cell cycle progression, cyclin E also has a role in regulating cell cycle arrest in response to DNA damage.

Artemis regulates cell cycle recovery from the S phase checkpoint by promoting degradation of cyclin E.

Artemis, a member of the SNM1 gene family, is a known phosphorylation target of ATM, ATR, and DNA-PKcs. We have previously identified two serine residues in Artemis (Ser(516) and Ser(645)) that are subject to phosphorylation by ATM and are involved in mediating recovery from the G(2)/M checkpoint in response to ionizing radiation. Here we show that these same sites are also phosphorylated by ATR in response to various types of replication stress including UVC, aphidicolin, and hydroxyurea. We also show that mutation of the Ser(516) and Ser(645) residues causes a prolonged S phase checkpoint recovery after treatment with UV or aphidicolin, and that this delayed recovery process coincides with a prolonged stabilization of cyclin E and down-regulation of Cdk2 kinase activity. Furthermore, we show that Artemis interacts with the F-box protein Fbw7, and that this interaction regulates cyclin E degradation through the SCF(Fbw7) E3 ubiquitin ligase complex. The interaction between Artemis and Fbw7 is regulated by phosphorylation of Ser(516) and Ser(645) sites that occur in response to replication stress. Thus, our findings suggest a novel pathway of recovery from the S phase checkpoint in that in response to replication stress phosphorylation of Artemis by ATR enhances its interaction with Fbw7, which in turn promotes ubiquitylation and the ultimate degradation of cyclin E.

Artemis links ATM to G2/M checkpoint recovery via regulation of Cdk1-cyclin B.

Artemis is a phospho-protein that has been shown to have roles in V(D)J recombination, nonhomologous end-joining of double-strand breaks, and regulation of the DNA damage-induced G(2)/M cell cycle checkpoint. Here, we have identified four sites in Artemis that are phosphorylated in response to ionizing radiation (IR) and show that ATM is the major kinase responsible for these modifications. Two of the sites, S534 and S538, show rapid phosphorylation and dephosphorylation, and the other two sites, S516 and S645, exhibit rapid and prolonged phosphorylation. Mutation of both of these latter two residues results in defective recovery from the G(2)/M cell cycle checkpoint. This defective recovery is due to promotion by mutant Artemis of an enhanced interaction between unphosphorylated cyclin B and Cdk1, which in turn promotes inhibitory phosphorylation of Cdk1 by the Wee1 kinase. In addition, we show that mutant Artemis prevents Cdk1-cyclin B activation by causing its retention in the centrosome and inhibition of its nuclear import during prophase. These findings show that ATM regulates G(2)/M checkpoint recovery through inhibitory phosphorylations of Artemis that occur soon after DNA damage, thus setting a molecular switch that, hours later upon completion of DNA repair, allows activation of the Cdk1-cyclin B complex. These findings thus establish a novel function of Artemis as a regulator of the cell cycle in response to DNA damage.

The multifunctional SNM1 gene family: not just nucleases.

The archetypical member of the SNM1 gene family was discovered 30 years ago in the budding yeast Saccharomyces cerevisiae. This small but ubiquitous gene family is characterized by metallo-beta-lactamase and beta-CASP domains, which together have been demonstrated to comprise a nuclease activity. Three mammalian members of this family, SNM1A, SNM1B/Apollo and Artemis, have been demonstrated to play surprisingly divergent roles in cellular metabolism. These pathways include variable (diversity) joining recombination, nonhomologous end-joining of double-strand breaks, DNA damage and mitotic cell cycle checkpoints, telomere maintenance and protein ubiquitination. Not all of these functions are consistent with a model in which these proteins act only as nucleases, and indicate that the SNM1 gene family encodes multifunctional products that can act in diverse biochemical pathways. In this article we discuss the various functions of SNM1A, SNM1B/Apollo and Artemis.

Double-strand breaks induce homologous recombinational repair of interstrand cross-links via cooperation of MSH2, ERCC1-XPF, REV3, and the Fanconi anemia pathway.

DNA interstrand cross-linking agents have been widely used in chemotherapeutic treatment of cancer. The majority of interstrand cross-links (ICLs) in mammalian cells are removed via a complex process that involves the formation of double-strand breaks at replication forks, incision of the ICL, and subsequent error-free repair by homologous recombination. How double-strand breaks effect the removal of ICLs and the downstream homologous recombination process is not clear. Here, we describe a plasmid-based recombination assay in which one copy of the CFP gene is inactivated by a site-specific psoralen ICL and can be repaired by gene conversion with a mutated homologous donor sequence. We found that the homology-dependent recombination (HDR) is inhibited by the ICL. However, when we introduced a double-strand break adjacent to the site of the ICL, the removal of the ICL was enhanced and the substrate was funneled into a HDR repair pathway. This process was not dependent on the nucleotide excision repair pathway, but did require the ERCC1-XPF endonuclease and REV3. In addition, both the Fanconi anemia pathway and the mismatch repair protein MSH2 were required for the recombinational repair processing of the ICL. These results suggest that the juxtaposition of an ICL and a DSB stimulates repair of ICLs through a process requiring components of mismatch repair, ERCC1-XPF, REV3, Fanconi anemia proteins, and homologous recombination repair factors.

SNM1A acts downstream of ATM to promote the G1 cell cycle checkpoint.

We have shown previously that SNM1A colocalizes with 53BP1 at sites of double-strand breaks (DSBs) induced by IR, and that these proteins interact with or without DNA damage. However, the role of SNM1A in the DNA damage response has not been elucidated. Here, we show that SNM1A is required for an efficient G1 checkpoint arrest after IR exposure. Interestingly, the localization of SNM1A to sites of DSBs does not require either 53BP1 or H2AX, nor does the localization of 53BP1 require SNM1A. However, the localization of SNM1A does require ATM. Furthermore, SNM1A is shown to be a phosphorylation substrate of ATM in vitro, and to interact with ATM in vivo particularly after exposure of cells to IR. In addition, in the absence of SNM1A the activation of the downstream ATM target p53 is reduced. These findings suggest that SNM1A acts with ATM to promote the G1 cell cycle checkpoint.

Snm1-deficient mice exhibit accelerated tumorigenesis and susceptibility to infection.

The eukaryotic SNM1 gene family has been implicated in a number of cellular pathways, including repair of DNA interstrand cross-links, involvement in VDJ recombination, repair of DNA double-strand breaks, and participation in cell cycle checkpoint pathways. In particular, mammalian SNM1 has been shown to be required in a mitotic checkpoint that causes arrest of cells in prophase prior to chromosome condensation in response to spindle poisons. Here, we report on the phenotype of a knockout of Snm1 in the mouse. Snm1-/- mice are viable and fertile but exhibit a complex phenotype. Both homozygous and heterozygous mice show a decline in survival compared to wild-type littermates. In homozygous mutant males, this reduction in survival is principally due to bacterial infections in the preputial and mandibular glands and to a lesser extent to tumorigenesis, while in homozygous and heterozygous females, it is due almost solely to tumorigenesis. The high incidence of bacterial infections in the homozygous mutant males suggests an immune dysfunction; however, examinations of T- and B-cell development and immunoglobulin class switching did not reveal a defect in these pathways. Crossing of Snm1 mutant mice with a Trp53 null mutant resulted in an increase in mortality and a restriction of the tumor type to lymphomas, particularly those of the thymus. Taken together, these findings demonstrate that Snm1 is a tumor suppressor in mice that in addition has a role in immunity.

Repair of DNA interstrand cross-links: interactions between homology-dependent and homology-independent pathways.

DNA interstrand cross-links (ICLs) are complex DNA lesions generated by bifunctional alkylating agents, a class of compounds extensively used in cancer chemotherapy. Formation of an ICL covalently links the opposing strands of the double helix and results in severe disruptions of normal DNA functions, such as replication, transcription, and recombination. Because of the structural complexity, ICLs are most likely recognized by a variety of repair recognition proteins and processed through multiple mechanisms. To study the involvement of different repair pathways in ICL processing, we examined a variety of mammalian mutants with distinct DNA repair deficiencies. We found that the presence of ICLs induces frequent recombination between direct repeat sequences, suggesting that the single-strand annealing pathway may be an important mechanism for the removal of ICLs situated within direct repeats. Unlike recombination-independent ICL repair, ICL-induced single-strand annealing does not require the nucleotide excision repair (NER) mechanism. In cells defective in the mismatch repair protein Msh2, the level of recombination-independent ICL repair was significantly increased, suggesting that processing by the mismatch repair mechanism may lead to recombinational repair of ICLs. Our results suggest that removal of ICLs may involve two error-prone mechanisms depending on the sequence context of the cross-linked site.

hMutSbeta is required for the recognition and uncoupling of psoralen interstrand cross-links in vitro.

The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutSbeta but is not dependent upon the hMutSalpha complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutSbeta complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.

Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response.

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.

Nucleotide excision repair- and polymerase eta-mediated error-prone removal of mitomycin C interstrand cross-links.

Interstrand cross-links (ICLs) make up a unique class of DNA lesions in which both strands of the double helix are covalently joined, precluding strand opening during replication and transcription. The repair of DNA ICLs has become a focus of study since ICLs are recognized as the main cytotoxic lesion inflicted by an array of alkylating compounds used in cancer treatment. As is the case for double-strand breaks, a damage-free homologous copy is essential for the removal of ICLs in an error-free manner. However, recombination-independent mechanisms may exist to remove ICLs in an error-prone fashion. We have developed an in vivo reactivation assay that can be used to examine the removal of site-specific mitomycin C-mediated ICLs in mammalian cells. We found that the removal of the ICL from the reporter substrate could take place in the absence of undamaged homologous sequences in repair-proficient cells, suggesting a cross-link repair mechanism that is independent of homologous recombination. Systematic analysis of nucleotide excision repair mutants demonstrated the involvement of transcription-coupled nucleotide excision repair and a partial requirement for the lesion bypass DNA polymerase eta encoded by the human POLH gene. From these observations, we propose the existence of a recombination-independent and mutagenic repair pathway for the removal of ICLs in mammalian cells.

hSnm1 colocalizes and physically associates with 53BP1 before and after DNA damage.

snm1 mutants of Saccharomyces cerevisiae have been shown to be specifically sensitive to DNA interstrand crosslinking agents but not sensitive to monofunctional alkylating agents, UV, or ionizing radiation. Five homologs of SNM1 have been identified in the mammalian genome and are termed SNM1, SNM1B, Artemis, ELAC2, and CPSF73. To explore the functional role of human Snm1 in response to DNA damage, we characterized the cellular distribution and dynamics of human Snm1 before and after exposure to DNA-damaging agents. Human Snm1 was found to localize to the cell nucleus in three distinct patterns. A particular cell showed diffuse nuclear staining, multiple nuclear foci, or one or two larger bodies confined to the nucleus. Upon exposure to ionizing radiation or an interstrand crosslinking agent, the number of cells exhibiting Snm1 bodies was reduced, while the population of cells with foci increased dramatically. Indirect immunofluorescence studies also indicated that the human Snm1 protein colocalized with 53BP1 before and after exposure to ionizing radiation, and a physical interaction was confirmed by coimmunoprecipitation assays. Furthermore, human Snm1 foci formed after ionizing radiation were largely coincident with foci formed by human Mre11 and to a lesser extent with those formed by BRCA1, but not with those formed by human Rad51. Finally, we mapped a region of human Snm1 of approximately 220 amino acids that was sufficient for focus formation when attached to a nuclear localization signal. Our results indicate a novel function for human Snm1 in the cellular response to double-strand breaks formed by ionizing radiation.

Deficiency in SNM1 abolishes an early mitotic checkpoint induced by spindle stress.

Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis. Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition. Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability. In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome. These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.

FANCI is a negative regulator of Akt activation.

Akt is a critical mediator of the oncogenic PI3K pathway, and its activation is regulated by kinases and phosphatases acting in opposition. We report here the existence of a novel protein complex that is composed minimally of Akt, PHLPP1, PHLPP2, FANCI, FANCD2, USP1 and UAF1. Our studies show that depletion of FANCI, but not FANCD2 or USP1, results in increased phosphorylation and activation of Akt. This activation is due to a reduction in the interaction between PHLPP1 and Akt in the absence of FANCI. In response to DNA damage or growth factor treatment, the interactions between Akt, PHLPP1 and FANCI are reduced consistent with the known phosphorylation of Akt in response to these stimuli. Furthermore, depletion of FANCI results in reduced apoptosis after DNA damage in accord with its role as a negative regular of Akt. Our findings describe an unexpected function for FANCI in the regulation of Akt and define a previously unrecognized intersection between the PI3K-Akt and FA pathways.

Translation of hSNM1 is mediated by an internal ribosome entry site that upregulates expression during mitosis.

SNM1 is involved in the repair of DNA interstrand cross-links (ICLs) in Saccharomyces cerevisiae and possibly in human cells, although relatively little is known about its biochemical function. The hSNM1 contains a long 5' untranslated region (5'UTR) predicted to fold into a complex secondary structure, and which contains numerous short open reading frames (ORFs). We show here using bicistronic constructs that human SNM1 mRNA contains an internal ribosome entry site (IRES) that generally suppresses translation, except during mitosis where translation is upregulated. These results suggest that hSNM1 may have a mitotic function possibly involved in response to DNA interstrand cross-linking agents.

UHRF1 contributes to DNA damage repair as a lesion recognition factor and nuclease scaffold.

We identified ubiquitin-like with PHD and RING finger domain 1 (UHRF1) as a binding factor for DNA interstrand crosslink (ICL) lesions through affinity purification of ICL-recognition activities. UHRF1 is recruited to DNA lesions in vivo and binds directly to ICL-containing DNA. UHRF1-deficient cells display increased sensitivity to a variety of DNA damages. We found that loss of UHRF1 led to retarded lesion processing and reduced recruitment of ICL repair nucleases to the site of DNA damage. UHRF1 interacts physically with both ERCC1 and MUS81, two nucleases involved in the repair of ICL lesions. Depletion of both UHRF1 and components of the Fanconi anemia (FA) pathway resulted in increased DNA damage sensitivity compared to defect of each mechanism alone. These results suggest that UHRF1 promotes recruitment of lesion-processing activities via its affinity to recognize DNA damage and functions as a nuclease recruitment scaffold in parallel to the FA pathway.

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27.

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double strand break repair, and stress-induced cell-cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27, and show that Cul4A-DDB1(DDB2-Artemis) regulates G1 phase cell cycle progression in mammalian cells.