Patients with alien hand syndrome (AHS) experience making apparently deliberate and purposeful movements with their hand against their will. However, the mechanisms contributing to these involuntary actions remain poorly understood. Here, we describe two experimental investigations in a patient with corticobasal syndrome (CBS) with alien hand behaviour in her right hand. First, we show that responses with the alien hand are made significantly more quickly to images of objects which afford an action with that hand compared to objects which afford an action with the unaffected hand. This finding suggests that involuntary grasping behaviours in AHS might be due to exaggerated, automatic motor activation evoked by objects which afford actions with that limb. Second, using a backwards masked priming task, we found normal automatic inhibition of primed responses in the patient's unaffected hand, but importantly there was no evidence of such suppression in the alien limb. Taken together, these findings suggest that grasping behaviours in AHS may result from exaggerated object affordance effects, which might potentially arise from disrupted inhibition of automatically evoked responses.
Alien limb; Object affordance; Automatic inhibition; Masked priming
The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells.
Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.
folate receptor alpha; FRA; ovarian cancer; fallopian tube; 1-carbon metabolism; reduced folate carrier; methylation
Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to regulate myriad intra-cellular processes. The addition and removal of ubiquitin or ubiquitin-like proteins from factors has recently been demonstrated as a key mechanism to modulate DNA damage response (DDR) pathways. It is thus, timely to evaluate the potential for ubiquitin pathway enzymes as DDR drug targets for therapeutic intervention. The synthetic lethal approach provides exciting opportunities for the development of targeted therapies to treat cancer: most tumours have lost critical DDR pathways, and thus rely more heavily on the remaining pathways, while normal tissues are still equipped with all DDR pathways. Here, we review key deubiquitylating enzymes (DUBs) involved in DDR pathways, and describe how targeting DUBs may lead to selective therapies to treat cancer patients.
Synthetic lethality; Ubiquitin; Deubiquitylating enzyme; DUB; DNA damage response; DNA repair; Drug discovery; Checkpoint control
cytokinesis; Dma1; Dma2; E3 ubiquitin-ligase; histone modification; RNF8; Septins; Septin-filament; ubiquitin
Previous work has established that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is stabilized in an ATM-dependent manner in response to DNA damage and acts as a cofactor for p53-mediated transcription. Here, we show that in response to DNA damage caused by ionizing radiation, hnRNP K is phosphorylated in an ATM-dependent manner. Furthermore, our data indicate that ATM-dependent hnRNP K phosphorylation is required for its stabilization and its function as a p53 transcriptional cofactor in response to DNA damage. These findings thereby establish hnRNP K as an ATM target and help define how ATM orchestrates p53-dependent transcriptional responses in response to genotoxic stress.
DNA damage; ATM; phosphorylation; transcription; p53
The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA damaging agents, thus suggesting an important link between RNA metabolism and DNA repair. Our results broaden the knowledge of DNA damage signaling networks and identify novel components of the DDR.
The conserved MRE11-RAD50-NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target for phosphorylation by cyclin-dependent kinases (CDKs). We show that NBS1 serine 432 (Ser-432) phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification of NBS1 stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of this phosphorylation not only negatively affects DSB repair by HR but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.
NBS1; cyclin-dependent kinase; homologous recombination; end joining; replication restart
DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and 2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and 2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and 2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and 2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.
DNA damage; DNA repair; DNA double-strand breaks; DNA-end resection; heterogeneous nuclear ribonucleoproteins
The Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of Mre11’s nuclease domains, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results reveal that Mre11 forms a flexible dimer and suggest that Nbs1 is not only a checkpoint adaptor, but also functionally impacts on Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1 interaction sites and weaken the Mre11–Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.
While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.
Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpjtm/tm) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpjtm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpjtm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.
Mutation of the gene CENPJ has been found to cause primary microcephaly, an inherited disorder that is characterised by severely reduced brain size. More recently, mutation of CENPJ has been associated with Seckel syndrome, a disorder that is characterised by a severe reduction in both brain and body size that is apparent at birth, mental retardation, and skeletal abnormalities, in addition to a number of other clinical manifestations. Here, we have generated a mouse that expresses only low levels of mouse Cenpj protein and find that it recapitulates many of the key features of Seckel syndrome. Moreover, we find that errors during the proliferation of Cenpjtm/tm cells frequently lead to abnormal numbers of chromosomes or damaged chromosomes, which is likely to be the cause of increased cell death during embryonic development and to contribute to the proportionate dwarfism that is characteristic of Seckel syndrome.
Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato.
ALC1, a novel PARP1-stimulated chromatin-remodelling enzyme promotes DNA repair.
Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here, we show that the G-quadruplex interacting drug pyridostatin promoted growth arrest in human cancer cells via inducing replication- and transcription-dependent DNA damage. Chromatin immunoprecipitation sequence (ChIP-Seq) analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage, and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein levels and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.
DNA double-strand breaks (DSBs), which are generated by ionizing radiation (IR) and a range of other DNA damaging agents, are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Previous studies have shown that NHEJ in Saccharomyces cerevisiae requires the Yku70p–Yku80p heterodimer and a complex consisting of DNA Ligase IV, Lif1p and Nej1p. Here, we report that Nej1p is phosphorylated in response to DNA damage in a manner that relies on the DNA damage checkpoint kinases Mec1p, Rad53p and Dun1p. By using a mutational approach, we have identified a consensus Dun1p phosphorylation site in Nej1p, and mutation of conserved serine residues within it leads to decreased NHEJ efficiency. These data, together with previous findings that Rad55p – a protein involved in HR – is phosphorylated analogously, point to there being a broad signalling network connecting DNA damage checkpoint responses with the regulation of DNA DSB repair activities.
S. cerevisiae; Checkpoint; DNA damage; DUN1; NHEJ; Phosphorylation
Post-translational modificationsof the histone octamer play important roles in regulating responses to DNA damage. Here, we reveal that Saccharomyces cerevisiae Rtt109p promotes genome stability and resistance to DNA damaging agents, and that it does this by functionally cooperating with the histone chaperone Asf1p to maintain normal chromatin structure. Furthermore, we show that, as for Asf1p, Rtt109p is required for histone H3 acetylation on lysine 56 (K56) in vivo. Moreover, we reveal that Rtt109p directly catalyzes this modification in vitro in a manner that is stimulated by Asf1p. These data establish Rtt109p as a member of a new class of histone acetyl-transferases, and show that its actions are critical for cell survival in the presence of DNA damage during S-phase.
Rad51 is a key enzyme involved in DNA double-strand break repair by homologous recombination. Here, we show that in response to DNA damage, budding yeast Rad51 is phosphorylated on Ser-192 in a manner that is primarily mediated by the DNA-damage responsive protein kinase Mec1. We show that mutating Rad51 Ser-192 to Ala or Glu confers hypersensitivity to DNA damage and homologous recombination defects. Furthermore, biochemical analyses indicate that Ser-192 is required for Rad51 ATP hydrolysis and DNA binding activity in vitro, while mutation of Ser-192 does not markedly interfere with Rad51 multimer formation. These data suggest a model in which Mec1-mediated phosphorylation of Rad51 Ser-192 in response to DNA damage controls Rad51 activity and DNA repair by homologous recombination.
DNA repair; homologous recombination; Mec1; phosphorylation; Rad51
DNA double-strand breaks (DSBs) are extremely cytotoxic lesions with a single unrepaired DSB being sufficient to induce cell death. A complex signaling cascade, termed the DNA damage response (DDR), is in place to deal with such DNA lesions and maintain genome stability. Recent work by us and others has found that the signaling cascade activated by DSBs in mitosis is truncated, displaying apical, but not downstream, components of the DDR. The E3 Ubiquitin ligases RNF8, RNF168 and BRCA1, along with the DDR mediator 53BP1, are not recruited to DSB sites in mitosis, and activation of downstream checkpoint kinases is also impaired. Here, we show that RNF8 and RNF168 are recruited to DNA damage foci in late mitosis, presumably to prime sites for 53BP1 recruitment in early G1. Interestingly, we show that, although RNF8, RNF168 and 53BP1 are excluded from DSB sites during most of mitosis, they associate with mitotic structures such as the kinetochore, suggesting roles for these DDR factors during mitotic cell division. We discuss these and other recent findings and suggest how these novel data collectively contribute to our understanding of mitosis and how cells deal with DNA damage during this crucial cell cycle stage.
mitosis; DNA damage response; DNA double-strand breaks; signaling cascade; chromatin
AI is unable to make eye-movements and has a deficit of reflexive attention (Smith, Rorden, & Jackson, 2004). Here, we demonstrate that despite these deficits AI exhibits Inhibition of Return (IOR) for peripherally cued objects and locations. These data suggest that an intact oculomotor system is not required for the generation of either object-based or location-based IOR, and are consistent with the view that the early, facilitatory effects of peripheral cues and late IOR effects are mediated by different mechanisms.
SIRT6 belongs to the sirtuin family of protein lysine deacetylases (KDACs) that regulates ageing and genome stability. Here, we report a role for human SIRT6 in promoting DNA-end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination (HR). SIRT6 depletion impairs the accumulation of replication protein A (RPA) and single-stranded DNA (ssDNA) at DNA-damage sites, reduces rates of HR and sensitises cells to DSB-inducing agents. We identify the DSB-resection protein CtIP as a SIRT6 interaction partner and show that SIRT6-dependent CtIP deacetylation promotes resection. A non-acetylatable CtIP mutant alleviates the effect of SIRT6 depletion on resection, thus identifying CtIP as a key substrate by which SIRT6 facilitates DSB processing and HR. These findings further define how SIRT6 promotes genome stability.
Several common neuropsychiatric disorders (e.g., obsessive-compulsive disorder, Tourette syndrome (TS), autistic spectrum disorder) are associated with unpleasant bodily sensations that are perceived as an urge for action. Similarly, many of our everyday behaviors are also characterized by bodily sensations that we experience as urges for action. Where do these urges originate? In this paper, we consider the nature and the functional anatomy of “urges-for-action,” both in the context of everyday behaviors such as yawning, swallowing, and micturition, and in relation to clinical disorders in which the urge-for-action is considered pathological and substantially interferes with activities of daily living (e.g., TS). We review previous frameworks for thinking about behavioral urges and demonstrate that there is considerable overlap between the functional anatomy of urges associated with everyday behaviors such as swallowing, yawning, and micturition, and those urges associated with the generation of tics in TS. Specifically, we show that the limbic sensory and motor regions—insula and mid-cingulate cortex—are common to all of these behaviors, and we argue that this “motivation-for-action” network should be considered distinct from an “intentional action” network, associated with regions of premotor and parietal cortex, which may be responsible for the perception of “willed intention” during the execution of goal-directed actions.
Urge; Urge-for-action; Habit; Insula; Tourette syndrome; Action
FLOWERING LOCUS T (FT) protein is known to be part of the mobile flowering inducing “florigen” signal in plants, but it may not be acting alone. This article reviews the data that FT mRNA can also move systemically throughout the plant and into the shoot apical meristem (SAM) independently of the FT protein. There is a promotion of flowering when increased levels of virally expressed FT mRNA are present together with endogenously produced FT protein in inducing conditions, even if the additional FT mRNA is non-translatable and thus not increasing the overall levels of FT protein. A specific sequence, or “zip code” of the FT mRNA is required for systemic movement and this sequence binds a specific protein(s) in plant extracts. This raises the possibility the FT mRNA may be moving systemically through the plant and into the SAM as an RNA–protein complex, whether FT protein is also a component of this mobile complex remains to be determined.
flowering locus T; FT; mRNA; flowering; tobacco
Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5) but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2). Here, we report two mutations in the CtIP (RBBP8) gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder.
Cellular DNA is frequently damaged through the actions of exogenous and endogenously arising DNA damaging agents. To maintain genome integrity, cells have evolved complex mechanisms to detect DNA damage, signal its presence, and mediate its repair. The importance of such mechanisms is evident because inherited defects in them can cause embryonic lethality or severe genetically inherited diseases. The clinical manifestations of such diseases are complex and include growth delay, mental retardation, skeletal abnormalities, and predisposition to cancer. While most such syndromes are inherited recessively, in some cases they are inherited dominantly. Here, we show that mutations in CtIP/RBBP8 cause related disorders: Seckel and Jawad syndromes. In addition to revealing how mutated CtIP impairs responses to DNA damage in Seckel cells, we establish that, despite the recessive mode of inheritance for this syndrome, the Seckel mutation has a dominant manifestation at the cellular level. To our knowledge, this represents a new form of molecular mechanism for recessive inheritance of a human disease. Furthermore, the aberrantly spliced mRNA is expressed at very low levels and yet significantly impairs cellular functions and causes severe clinical symptoms. This should provide new awareness that even very subtle splice mutations may have pronounced pathogenic potential.