Redox regulation of RAD51 Cys319 and homologous recombination by peroxiredoxin 1.

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.

Aggresome assembly at the centrosome is driven by CP110-CEP97-CEP290 and centriolar satellites.

Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells. Centriolar satellites are proteinaceous granules implicated in the trafficking of proteins to the centrosome. During aggresome assembly, satellites were required for the growth of the aggresomal structure from an initial ring of phosphorylated HSP27 deposited around the centrioles. The seeding of this phosphorylated HSP27 ring depended on the centrosomal proteins CP110, CEP97 and CEP290. Owing to limiting amounts of CP110, senescent cells, which are characterized by the accumulation of protein aggregates, were defective in aggresome formation. Furthermore, satellites and CP110-CEP97-CEP290 were required for the aggregation of mutant huntingtin. Together, these data reveal roles for CP110-CEP97-CEP290 and satellites in the control of cellular proteostasis and the aggregation of disease-relevant proteins.

Primary cilia and the DNA damage response: linking a cellular antenna and nuclear signals.

The maintenance of genome stability involves integrated biochemical activities that detect DNA damage or incomplete replication, delay the cell cycle, and direct DNA repair activities on the affected chromatin. These processes, collectively termed the DNA damage response (DDR), are crucial for cell survival and to avoid disease, particularly cancer. Recent work has highlighted links between the DDR and the primary cilium, an antenna-like, microtubule-based signalling structure that extends from a centriole docked at the cell surface. Ciliary dysfunction gives rise to a range of complex human developmental disorders termed the ciliopathies. Mutations in ciliopathy genes have been shown to impact on several functions that relate to centrosome integrity, DNA damage signalling, responses to problems in DNA replication and the control of gene expression. This review covers recent findings that link cilia and the DDR and explores the various roles played by key genes in these two contexts. It outlines how proteins encoded by ciliary genes impact checkpoint signalling, DNA replication and repair, gene expression and chromatin remodelling. It discusses how these diverse activities may integrate nuclear responses with those that affect a structure of the cell periphery. Additional directions for exploration of the interplay between these pathways are highlighted, with a focus on new ciliary gene candidates that alter genome stability.

Altered gene regulation as a candidate mechanism by which ciliopathy gene SDCCAG8 contributes to schizophrenia and cognitive function.

Mutations in genes that encode centrosomal/ciliary proteins cause severe cognitive deficits, while common single-nucleotide polymorphisms in these genes are associated with schizophrenia (SZ) and cognition in genome-wide association studies. The role of these genes in neuropsychiatric disorders is unknown. The ciliopathy gene SDCCAG8 is associated with SZ and educational attainment (EA). Genome editing of SDCCAG8 caused defects in primary ciliogenesis and cilium-dependent cell signalling. Transcriptomic analysis of SDCCAG8-deficient cells identified differentially expressed genes that are enriched in neurodevelopmental processes such as generation of neurons and synapse organization. These processes are enriched for genes associated with SZ, human intelligence (IQ) and EA. Phenotypic analysis of SDCCAG8-deficent neuronal cells revealed impaired migration and neuronal differentiation. These data implicate ciliary signalling in the aetiology of SZ and cognitive dysfunction. We found that centrosomal/ciliary genes are enriched for association with IQ, suggesting altered gene regulation as a general model for neurodevelopmental impacts of centrosomal/ciliary genes.

Centrobin controls primary ciliogenesis in vertebrates.

The BRCA2 interactor, centrobin, is a centrosomal protein that has been implicated in centriole duplication and microtubule stability. We used genome editing to ablate CNTROB in hTERT-RPE1 cells and observed an increased frequency of monocentriolar and acentriolar cells. Using a novel monoclonal antibody, we found that centrobin primarily localizes to daughter centrioles but also associates with mother centrioles upon serum starvation. Strikingly, centrobin loss abrogated primary ciliation upon serum starvation. Ultrastructural analysis of centrobin nulls revealed defective axonemal extension after mother centriole docking. Ciliogenesis required a C-terminal portion of centrobin that interacts with CP110 and tubulin. We also depleted centrobin in zebrafish embryos to explore its roles in an entire organism. Centrobin-depleted embryos showed microcephaly, with curved and shorter bodies, along with marked defects in laterality control, morphological features that indicate ciliary dysfunction. Our data identify new roles for centrobin as a positive regulator of vertebrate ciliogenesis.

Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes.

Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression.

Centrosomes in the DNA damage response--the hub outside the centre.

Here, we review how DNA damage affects the centrosome and how centrosomes communicate with the DNA damage response (DDR) apparatus. We discuss how several proteins of the DDR are found at centrosomes, including the ATM, ATR, CHK1 and CHK2 kinases, the BRCA1 ubiquitin ligase complex and several members of the poly(ADP-ribose) polymerase family. Stereotypical centrosome organisation, in which two centriole barrels are orthogonally arranged in a roughly toroidal pericentriolar material (PCM), is strongly affected by exposure to DNA-damaging agents. We describe the genetic dependencies and mechanisms for how the centrioles lose their close association, and the PCM both expands and distorts after DNA damage. Another consequence of genotoxic stress is that centrosomes undergo duplication outside the normal cell cycle stage, meaning that centrosome amplification is commonly seen after DNA damage. We discuss several potential mechanisms for how centrosome numbers become dysregulated after DNA damage and explore the links between the DDR and the PLK1- and separase-dependent mechanisms that drive centriole separation and reduplication. We also describe how centrosome components, such as centrin2, are directly involved in responding to DNA damage. This review outlines current questions on the involvement of centrosomes in the DDR.

Protein stability versus function: effects of destabilizing missense mutations on BRCA1 DNA repair activity.

Mutations in breast cancer susceptibility gene BRCA1 (breast cancer early-onset 1) are associated with increased risk of developing breast and ovarian cancers. BRCA1 is a large protein of 1863 residues with two small structured domains at its termini: a RING domain at the N-terminus and a BRCT (BRCA1 C-terminus domain) repeat domain at the C-terminus. Previously, we quantified the effects of missense mutations on the thermodynamic stability of the BRCT domains, and we showed that many are so destabilizing that the folded functional state is drastically depopulated at physiological temperature. In the present study, we ask whether and how reduced thermodynamic stability of the isolated BRCT mutants translates into loss of function of the full-length protein in the cell. We assessed the effects of missense mutants on different stages of BRCA1-mediated DNA repair by homologous recombination using chicken lymphoblastoid DT40 cells as a model system. We found that all of the mutations, regardless of how profound their destabilizing effects, retained some DNA repair activity and thereby partially rescued the chicken BRCA1 knockout. By contrast, the mutation R1699L, which disrupts the binding of phosphorylated proteins (but which is not destabilizing), was completely inactive. It is likely that both protein context (location of the BRCT domains at the C-terminus of the large BRCA1 protein) and cellular environment (binding partners, molecular chaperones) buffer these destabilizing effects such that at least some mutant protein is able to adopt the folded functional state.

Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.

Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification.

Centrosome duplication is licensed by the disengagement, or 'uncoupling', of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the anaphase-promoting complex (APC/C), leading to securin degradation and release of active separase. Although APC/C activation during G2 arrest is dependent on polo-like kinase 1 (Plk1)-mediated degradation of the APC/C inhibitor, early mitotic inhibitor 1 (Emi1), Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulates centriole disengagement in response to hydroxyurea or DNA damage-induced cell-cycle arrest and this leads to centrosome amplification. However, the reduplication of disengaged centrioles is dependent on cyclin-dependent kinase 2 (Cdk2) activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Although release from these arrests leads to mitotic entry, the presence of disengaged and/or amplified centrosomes results in the formation of abnormal mitotic spindles that lead to chromosome mis-segregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability.

A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.

DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis.

Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress caused by cellular respiration and DNA replication. We demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced prosurvival gene expression and strong activation of p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. Our results demonstrate that quiescence is a double-edged sword that renders HSCs intrinsically vulnerable to mutagenesis following DNA damage.

Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells.

The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.

Centriole separation in DNA damage-induced centrosome amplification.

Altered centrosome numbers are seen in tumor cells in response to DNA damaging treatments and are hypothesised to contribute to cancer development. The mechanism by which the centrosome and chromosome cycles become disconnected after DNA damage is not yet clear. Here, we show that centrosome amplification occurs after ionising radiation (IR) in chicken DT40 cells that lack DNA-PK, Ku70, H2AX, Xpa, and Scc1, demonstrating that these activities are not required for centrosome amplification. We show that inhibition of topoisomerase II induces Chk1-dependent centrosome amplification, a similar response to that seen after IR. In the immortalised, nontransformed hTERT-RPE1 line, we observed centriole splitting, followed by dose-dependent centrosome amplification, after IR. We found that IR results in the formation of single, not multiple, daughter centrioles during centrosome amplification in U2OS osteosarcoma cells. Analysis of BRCA1 and BRCA2 mutant tumor cells showed high levels of centriole splitting in the absence of any treatment. IR caused pronounced levels of centrosome amplification in BRCA1 mutant breast cancer cells. These data show that centrosome amplification occurs after different forms of DNA damage in chicken cells, in nontransformed human cells and in human tumor cell lines, indicating that this is a general response to DNA damaging treatments. Together, our data suggest that centriole splitting is a key step in potentiation of the centrosome amplification that is a general response to DNA damage.

Ku70 prevents genome instability resulting from heterozygosity of the telomerase RNA component in a vertebrate tumour line.

Telomere repeat sequences are added to linear chromosome ends by telomerase, an enzyme comprising a reverse transcriptase (TERT) and an RNA template component (TR). We aimed to investigate TR in the DT40 B-cell tumour line using gene targeting, but were unable to generate TR nulls, suggesting a requirement for TR in DT40 proliferation. Disruption of one TR allele reduced telomerase activity and caused a progressive decline in telomere and G-strand overhang length. We then examined the interactions between TR and cellular DNA double-strand break (DSB) repair. Deletion in TR+/- cells of the gene encoding the non-homologous end-joining protein, Ku70, caused rapid loss of G-strand overhangs. Ku70-/-TR+/- cells proliferated more slowly than either single mutant and showed frequent mitotic aberrations. Activation of the DNA damage response was observed in TR-deficient cells and was exacerbated by Ku deficiency, although frequent telomeric DNA damage signals were not observed until late passages. This activation of the DNA damage response was suppressed by deletion of Rad54, a key homologous recombination gene. These findings suggest that Ku and telomerase cooperate to block homologous recombination from acting on telomeres.

DNA damage induces Chk1-dependent centrosome amplification.

Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM (ataxia telangiectasia, mutated)- and ATR (ATM and Rad3-related)-defective cells, indicating a complementary role for these DNA-damage-responsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 (Chk1) by RNA-mediated interference or drug treatment suppressed DNA-damage-induced centrosome amplification. Radiation-induced centrosome amplification was abrogated in Chk1(-/-) DT40 cells, but occurred at normal levels in Chk1(-/-) cells transgenically expressing Chk1. Expression of kinase-dead Chk1, or Chk1S345A, through which the phosphatidylinositol-3-kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.

Involvement of centrosome amplification in radiation-induced mitotic catastrophe.

Cells exposed to ionizing radiation die via different mechanisms, including apoptosis and mitotic catastrophe. To determine the frequency of mitotic catastrophe in tumor cells after irradiation, we used time-lapse imaging to track centrin-1 and histone H2B in U2OS osteosarcoma cells. We observed a dose-dependent increase in the frequency of mitotic catastrophe after irradiation, although a consistent 30% of cell death occurred through mitotic failure at doses from 2-10 Gy. One potential cause of mitotic catastrophe is centrosome amplification, which is induced by irradiation, and which can result in the formation of multipolar mitotic spindles. Up to 60% of mitotic catastrophes occurred in cells with >2 centrosomes after irradiation. We observed multipolar mitoses in p53(+) and p53(-) tumor cells after irradiation and found that the spindle assembly checkpoint is active in multipolar mitotic cells. However, we did not detect active caspase-3 in multipolar mitoses. These data demonstrate that a significant proportion of cell death induced by ionizing irradiation is through an apoptosis-independent mechanism involving centrosome amplification and mitotic catastrophe.