: Human progeroid Werner syndrome provides the current best model for analysis of human aging, recapitulating many aspects of normal aging as a result of mutation of the WRN gene. This gene encodes a RecQ‐type helicase with additional... more
: Human progeroid Werner syndrome provides the current best model for analysis of human aging, recapitulating many aspects of normal aging as a result of mutation of the WRN gene. This gene encodes a RecQ‐type helicase with additional exonuclease activity. While biochemical studies in vitro have proven invaluable in determining substrate specificities of the WRN exonuclease and helicase, it has been difficult to dissociate the two key enzyme activities in vivo. We are developing Drosophila as a model system for analysis of WRN function; the suitability of Drosophila for extensive and sophisticated genetic manipulation permits us to investigate regulatory pathways and the impact of WRN loss at organismal, cellular, and molecular levels. BLASTP screening of the Drosophila genome with human WRN sequence allowed us to identify three RecQ helicases with strong homology to human WRN, a presumed helicase component of the spliceosome, and two DEAH‐box putative RNA helicases with weaker WRN homology. None of these helicases contain a WRN‐like exonuclease domain, but two potential WRN‐like exonucleases in flies encoded by the loci CG7670 and CG6744 were also identified in the BLAST search. CG6744 and CG7670 are more closely related to human WRN than to each other. We have obtained a fly strain with a piggyBac insertional mutation within the CG6744 locus, which decreases expression of the encoded mRNA. Such flies show elevated levels of somatic recombination. We suggest that WRN‐like exonuclease activity is critical in maintaining genomic integrity in flies.
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The human tumour suppressor protein p53 is critical for regulation of the cell cycle on genotoxic insult. When DNA is damaged by radiation, chemicals or viral infection, cells respond rapidly by arresting the cell cycle. A G1 arrest... more
The human tumour suppressor protein p53 is critical for regulation of the cell cycle on genotoxic insult. When DNA is damaged by radiation, chemicals or viral infection, cells respond rapidly by arresting the cell cycle. A G1 arrest requires the activity of wild‐type p53, as it is not observed in cells lacking functionally wild‐type protein, and at least some component of S phase and G2/M arrests is also thought to be p53‐dependent. p53 functions as a transcription factor which binds specific DNA sequences, and recently major downstream targets have been identified, including p21Cip1 an inhibitor of the cell cycle kinases that also blocks the replicative but not the repair function of DNA polymerase δ auxiliary factor, PCNA. Current interest focuses on developing novel cancer therapies based on our knowledge of the activity of p53 and p21Cip1 in the cell cycle.
Research Interests: Biology, Cell Cycle, Cell Biology, DNA damage, DNA repair, and 15 moreMedicine, Biological Sciences, Humans, Protein Kinases, Alternative splicing, Phosphorylation, Transcription Factor, Kinase, Oncogenes, Homeostasis, Alternative Splicing, Suppressor, Protein Biosynthesis, Psychology and Cognitive Sciences, and Medical and Health Sciences
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WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS),... more
WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.
Research Interests: Flow Cytometry, Biology, Cell Cycle, DNA replication, DNA damage, and 15 moreDNA repair, Medicine, Cell line, Humans, Biogerontology, Clinical Sciences, Comet Assay, Helicase, Camptothecin, Base Sequence, Chromosomal Instability, Hammerhead Ribozyme, Molecular Sequence Data, Genome Instability, and premature aging
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DNA replication is absolutely dependent upon opening up of the duplex template through the action of DNA-dependent helicases that utilise the energy from ATP hydrolysis to break hydrogen bonds between complementary base pairs (see Chapter... more
DNA replication is absolutely dependent upon opening up of the duplex template through the action of DNA-dependent helicases that utilise the energy from ATP hydrolysis to break hydrogen bonds between complementary base pairs (see Chapter 1). Such helicases must be able to bind to DNA at the replica...
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The premature human ageing... more
The premature human ageing Werner's syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670, with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3'-5' exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila, we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability.
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SummaryHuman aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the... more
SummaryHuman aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.
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Research Interests: Biology, DNA replication, Drosophila melanogaster, DNA repair, Medicine, and 12 moreWestern blotting, Humans, Animals, Oncogene, Clinical Sciences, Schizosaccharomyces Pombe, Genomic instability, Amino Acid Profile, Amino Acid Sequence, Recombinant Proteins, Dna Synthesis, and Molecular Sequence Data
Research Interests: Genetics, Biology, Cell Cycle, DNA replication, Cancer Prevention, and 15 moreMedicine, Biological Sciences, Humans, Escherichia coli, Cell Cycle regulation, Biological activity, Amino Acid Sequence, Recombinant Proteins, Protein Kinase Inhibitors, In Vitro Transcription, Molecular Sequence Data, Restriction Mapping, binding sites, Psychology and Cognitive Sciences, and Medical and Health Sciences
Research Interests: Biology, DNA replication, DNA repair, Medicine, Biological Sciences, and 15 moreDrosophila, Humans, Age, Animals, Phenotype, Helicase, Gene Expression Regulation, single stranded DNA, Werner Syndrome, RecQ Helicases, Genome Instability, Psychology and Cognitive Sciences, premature aging, Uracil, and Medical and Health Sciences
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Research Interests: Genetics, Biology, Drosophila melanogaster, Gene expression, Biological Sciences, and 15 moreHumans, Mutation, Animals, Helicase, Cancer Incidence, Genomic instability, Protein Conformation, Homologous Recombination, Camptothecin, Amino Acid Sequence, RecQ Helicases, Molecular Sequence Data, Genome Instability, alleles, and Medical and Health Sciences
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Bone is a complex organ serving roles in skeletal support and movement, and is a source of blood cells including adaptive and innate immune cells. Structural and functional integrity is maintained through a balance between bone synthesis... more
Bone is a complex organ serving roles in skeletal support and movement, and is a source of blood cells including adaptive and innate immune cells. Structural and functional integrity is maintained through a balance between bone synthesis and bone degradation, dependent in part on mechanical loading but also on signaling and influences of the tissue microenvironment. Bone structure and the extracellular bone milieu change with age, predisposing to osteoporosis and increased fracture risk, and this is exacerbated in patients with diabetes. Such changes can include loss of bone mineral density, deterioration in micro-architecture, as well as decreased bone flexibility, through alteration of proteinaceous bone support structures, and accumulation of senescent cells. Senescence is a state of proliferation arrest accompanied by marked morphological and metabolic changes. It is driven by cellular stress and serves an important acute tumor suppressive mechanism when followed by immune-media...
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The nuclear envelope (NE) is a highly complex dynamic structure that must provide a boundary separating the cytoplasm from the nucleoplasm during inter-phase but not during mitosis, when chromosomes are segregated between two daughter... more
The nuclear envelope (NE) is a highly complex dynamic structure that must provide a boundary separating the cytoplasm from the nucleoplasm during inter-phase but not during mitosis, when chromosomes are segregated between two daughter cells. In other words, the nuclear envelope must be stable during interphase but unstable during mitosis. Understanding the processes that control nuclear envelope breakdown (NEBD) and reassembly has been the subject of intensive research during the past eight years, such that over the last year or two a consensus has started to emerge that was put succinctly by John Newport at the 1992 Lancaster symposium of the Society of Experimental Biology, when he expressed the view that NEBD and reassembly are extremes of a dynamic process involving continuous structural rearrangements of the components of the envelope. In this chapter we will review what is currently known about this process and propose a general model for the regulation of the major events involved.
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Abstract: The premature ageing Werner syndrome (WS) is characterized by the early onset of many age related pheno-types, including graying of hair, cataracts, atherosclerosis, cancer and type 2 diabetes. Type 2 diabetes (DM2) is the loss... more
Abstract: The premature ageing Werner syndrome (WS) is characterized by the early onset of many age related pheno-types, including graying of hair, cataracts, atherosclerosis, cancer and type 2 diabetes. Type 2 diabetes (DM2) is the loss of blood glucose homeostastis, due to insulin resistance and a failure of acute glucose-stimulated insulin secretion (GSIS) by pancreatic cells. Early compensation for insulin resistance usually occurs through increased insulin secretion conse-quent on increased cell mass, requiring proliferation of cells; DM2 progresses if there is failure of compensation. How loss of the WRN DNA helicase/exonuclease in WS contributes to DM2 has long been a puzzle. Loss of function muta-tions in WRN result in problems with DNA replication, repair and recombination, consequential genomic instability and premature onset of cellular senescence. Here, I suggest that the high prevalence of DM2 in WS is a consequence of senes-cence of WS cells, with islet cells undergoin...
Acute inflammation is a physiological response to injury or infection, with a cascade of steps that ultimately leads to recruitment of immune cells to clear invading pathogens and heal wounds. However, chronic inflammation arising from... more
Acute inflammation is a physiological response to injury or infection, with a cascade of steps that ultimately leads to recruitment of immune cells to clear invading pathogens and heal wounds. However, chronic inflammation arising from continued presence of the initial trigger, or dysfunction of signalling and/or effector pathways, is harmful to health. While successful ageing in older adults including centenarians is associated with low levels of inflammation, elevated inflammation increases the risk of poor health and death [1–3]. Hence inflammation has been described as one of seven pillars of ageing. Age-associated sterile, chronic, and low-grade inflammation is commonly termed inflammageing - it is not simply a consequence of increasing chronological age, but is also a marker of biological ageing, multimorbidity and mortality risk. While inflammageing was initially thought to be caused by “continuous antigenic load and stress”, reports from the last two decades describe a much ...
Research Interests: Immunology, Senescence, Inflammation, Medicine, Immune system, and 3 moreCells, Immunosenescence, and Ageing
Drugs that remove senescent cells cut coronavirus deaths in old mice
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DNA replication is absolutely dependent upon opening up of the duplex template through the action of DNA-dependent helicases that utilise the energy from ATP hydrolysis to break hydrogen bonds between complementary base pairs (see Chapter... more
DNA replication is absolutely dependent upon opening up of the duplex template through the action of DNA-dependent helicases that utilise the energy from ATP hydrolysis to break hydrogen bonds between complementary base pairs (see Chapter 1). Such helicases must be able to bind to DNA at the replica...
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Nucleases and helicases are involved in numerous steps in DNA replication and repair. Nucleases act on intermediates in DNA replication created by DNA polymerases (Chapter 4) and helicases (Chapter 3). They can create substrates for... more
Nucleases and helicases are involved in numerous steps in DNA replication and repair. Nucleases act on intermediates in DNA replication created by DNA polymerases (Chapter 4) and helicases (Chapter 3). They can create substrates for repair as in Okazaki fragment processing (OFP) and homologous recombination. They can also create substrates for activation of a checkpoint response, or participate in downregulation of checkpoints. In the special case of telomere replication, they are also involved in essential processing steps (Chapter 8). Nucleases known to act during DNA replication include Dna2, Rad27, Mre11, Sae2, Exo1, RNaseH, Yen1 andMus81/Mms4. Of these, Dna2, Exo1 and Mre11 are of particular interest because they have been identified as crucial activities that initiate repair of double-strand breaks (DSBs) by homologous recombination and thus form an intrinsic link between DNA replication and repair of DSBs derived from replication fork failure. The action of the nucleases is coordinated with those of a number of helicases and is discussed here in the context of a network of their interactions that combine to maintain genome integrity during DNA replication.
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Human aging is associated with loss of tissue homeostasis and a greatly elevated risk of debilitating disease, with associated costs in terms of diminished quality of life for the individual and financial burdens on health-care providers.... more
Human aging is associated with loss of tissue homeostasis and a greatly elevated risk of debilitating disease, with associated costs in terms of diminished quality of life for the individual and financial burdens on health-care providers. Any advances that hold out the realistic prospect of tackling age-related morbidity are therefore to be welcomed. The recent report by Jeskelioff et al. published in 2010 that telomerase reactivation in adult male mice can not only halt but actually reverse significant tissue pathologies provides a long-awaited proof of principle that it is possible to rejuvenate aged tissues. This review assesses the impact of this new work and considers possible problems and potential benefits of telomerase activation as a therapy to tackle the morbidities associated with human aging.
Research Interests: Aging, Biology, Medicine, Telomerase, Humans, and 3 moreClinical Sciences, Rejuvenation, and Telomere
Research Interests: Genetics, Biology, Cell Cycle, DNA replication, Cancer Prevention, and 15 moreMedicine, Biological Sciences, Humans, Escherichia coli, Cell Cycle regulation, Biological activity, Amino Acid Sequence, Recombinant Proteins, Protein Kinase Inhibitors, In Vitro Transcription, Molecular Sequence Data, Restriction Mapping, binding sites, Psychology and Cognitive Sciences, and Medical and Health Sciences
Bacteriophage lambda DNA is assembled into pseudonuclei in Xenopus egg extract and replicated semiconservatively under temporal cell cycle control. Here, replication is shown to be regulated spatially and to occur at discrete sites that... more
Bacteriophage lambda DNA is assembled into pseudonuclei in Xenopus egg extract and replicated semiconservatively under temporal cell cycle control. Here, replication is shown to be regulated spatially and to occur at discrete sites that represent clustered replication forks. Clustered forks are visualized by incorporation of biotin-19-dUTP into nascent DNA. Pulse-label experiments show that discrete replication foci persist throughout S phase. We conclude that the organization of replication forks into discrete clusters, previously described in eukaryotic nuclei, is not dependent on specific eukaryotic DNA sequences or on a preexisting inherited chromosomal or nuclear structure.
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Research Interests: Biology, DNA replication, DNA repair, Medicine, Biological Sciences, and 14 moreDrosophila, Humans, Age, Animals, Phenotype, Helicase, Gene Expression Regulation, Werner Syndrome, RecQ Helicases, Genome Instability, Psychology and Cognitive Sciences, premature aging, Uracil, and Medical and Health Sciences
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Research Interests: Biology, DNA replication, Drosophila melanogaster, DNA repair, Medicine, and 12 moreWestern blotting, Humans, Animals, Oncogene, Clinical Sciences, Schizosaccharomyces Pombe, Genomic instability, Amino Acid Profile, Amino Acid Sequence, Recombinant Proteins, Dna Synthesis, and Molecular Sequence Data
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Chronological age represents the greatest risk factor for many life9 threatening diseases including neurodegeneration, cancer and cardiovascular disease; 10 ageing also increases susceptibility to infectious disease. Current therapies... more
Chronological age represents the greatest risk factor for many life9 threatening diseases including neurodegeneration, cancer and cardiovascular disease; 10 ageing also increases susceptibility to infectious disease. Current therapies that 11 effectively tackle individual diseases may have little impact on the overall healthspan 12 of older individuals, who would still be vulnerable to other age-related pathologies. 13 However, recent progress in ageing research has highlighted the accumulation of 14 senescent cells with chronological age as a probable underlying cause of pathological 15 ageing. Cellular senescence is an essentially irreversible proliferation arrest 16 mechanism that has important roles in development, wound healing and preventing 17 cancer, but it may limit tissue function and cause widespread inflammation with age. 18 The serine/threonine kinase mTOR is a regulatory nexus heavily implicated in both 19 ageing and senescence. Excitingly, a growing body of research h...
Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage, and oncogene induction. While beneficial as an acute response to stress, the accumulation of... more
Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage, and oncogene induction. While beneficial as an acute response to stress, the accumulation of senescent cells with increasing age is thought to contribute adversely to the development of cancer and a number of other age-related diseases, including neurodegenerative diseases for which there are currently no effective disease-modifying therapies. Non-cell-autonomous effects of senescent cells have been suggested to arise through the SASP, a wide variety of proinflammatory cytokines, chemokines, and exosomes secreted by senescent cells. Here, we report an additional means of cell communication utilised by senescent cells via large numbers of membrane-bound intercellular bridges—or tunnelling nanotubes (TNTs)—containing the cytoskeletal components actin and tubulin, which form direct physical connections between cells. We observe the presence of ...
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Ageing, and particularly the onset of age-related diseases, is associated with tissue dysfunction and macromolecular damage, some of which can be attributed to accumulation of oxidative damage. Polyphenolic natural products such as... more
Ageing, and particularly the onset of age-related diseases, is associated with tissue dysfunction and macromolecular damage, some of which can be attributed to accumulation of oxidative damage. Polyphenolic natural products such as stilbenoids, flavonoids and chalcones have been shown to be effective at ameliorating several age-related phenotypes, including oxidative stress, inflammation, impaired proteostasis and cellular senescence, both in vitro and in vivo. Here we aim to identify the structural basis underlying the pharmacology of polyphenols towards ROS and related biochemical pathways involved in age-related disease. We compile and describe SAR trends across different polyphenol chemotypes including stilbenoids, flavonoids and chalcones, review their different molecular targets and indications, and identify common structural ground between chemotypes and mechanisms of action. In particular, we focus on the structural requirements for the direct scavenging of reactive oxygen/n...