Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.

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.

Novel DNA mismatch-repair activity involving YB-1 in human mitochondria.

Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.

Gene-specific and mitochondrial repair of oxidative DNA damage.

The Southern blot gene-specific DNA damage and repair assay is a robust and flexible method for quantifying many kinds of induced damage and repair with high reproducibility. Specific nicking and loss of a restricted DNA fragment at the site of induced damage is visualized by Southern blot and quantified against a control; since the blot is gene specific, only the damage of interest is measured. Here we show how the assay may be adapted to assess mitochondrial DNA (mtDNA) damage. In the mitochondrion, 8-oxoguanine is a significant oxidative lesion; in the laboratory, photoactivated methylene blue may be used to introduce this lesion into cells. Other lesions may also be studied by using different DNA damaging agents. We find that damage induction by methylene blue is consistently far greater in the mitochondrion than the nucleus. Thus advantageously, mitochondrial 8-oxoguanine repair may be studied without mtDNA isolation or preparation, which are processes known to induce DNA damage and skew measurements. This chapter gives detailed instructions for using methylene blue and the gene-specific repair assay to accurately measure mitochondrial oxidative damage and repair rates.

Mismatch repair activity in mammalian mitochondria.

Mitochondrial DNA (mtDNA) defects cause debilitating metabolic disorders for which there is no effective treatment. Patients suffering from these diseases often harbour both a wild-type and a mutated subpopulation of mtDNA, a situation termed heteroplasmy. Understanding mtDNA repair mechanisms could facilitate the development of novel therapies to combat these diseases. In particular, mismatch repair activity could potentially be used to repair pathogenic mtDNA mutations existing in the heteroplasmic state if heteroduplexes could be generated. To date, however, there has been no compelling evidence for such a repair activity in mammalian mitochondria. We now report evidence consistent with a mismatch repair capability in mammalian mitochondria that exhibits some characteristics of the nuclear pathway. A repair assay utilising a nicked heteroduplex substrate with a GT or a GG mismatch in the beta-galactosidase reporter gene was used to test the repair potential of different lysates. A low level repair activity was identified in rat liver mitochondrial lysate that showed no strand bias. The activity was mismatch-selective, bi-directional, ATP-dependent and EDTA-sensitive. Western analysis using antibody to MSH2, a key nuclear mismatch repair system (MMR) protein, showed no cross-reacting species in mitochondrial lysate. A hypothesis to explain the molecular mechanism of mitochondrial MMR in the light of these observations is discussed.

A fluorescence-based exonuclease assay to characterize DmWRNexo, orthologue of human progeroid WRN exonuclease, and its application to other nucleases.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

The Drosophila orthologue of progeroid human WRN exonuclease, DmWRNexo, cleaves replication substrates but is inhibited by uracil or abasic sites : analysis of DmWRNexo activity in vitro.

Werner syndrome (WS) is a rare late-onset premature ageing disease showing many of the phenotypes associated with normal ageing, and provides one of the best models for investigating cellular pathways that lead to normal ageing. WS is caused by mutation of WRN, which encodes a multifunctional DNA replication and repair helicase/exonuclease. To investigate the role of WRN protein's unique exonuclease domain, we have recently identified DmWRNexo, the fly orthologue of the exonuclease domain of human WRN. Here, we fully characterise DmWRNexo exonuclease activity in vitro, confirming 3'-5' polarity, demonstrating a requirement for Mg(2+), inhibition by ATP, and an ability to degrade both single-stranded DNA and duplex DNA substrates with 3' or 5' overhangs, or bubble structures, but with no activity on blunt ended DNA duplexes. We report a novel active site mutation that ablates enzyme activity. Lesional substrates containing uracil are partially cleaved by DmWRNexo, but the enzyme pauses on such substrates and is inhibited by abasic sites. These strong biochemical similarities to human WRN suggest that Drosophila can provide a valuable experimental system for analysing the importance of WRN exonuclease in cell and organismal ageing.

The role of DNA exonucleases in protecting genome stability and their impact on ageing.

Exonucleases are key enzymes involved in many aspects of cellular metabolism and maintenance and are essential to genome stability, acting to cleave DNA from free ends. Exonucleases can act as proof-readers during DNA polymerisation in DNA replication, to remove unusual DNA structures that arise from problems with DNA replication fork progression, and they can be directly involved in repairing damaged DNA. Several exonucleases have been recently discovered, with potentially critical roles in genome stability and ageing. Here we discuss how both intrinsic and extrinsic exonuclease activities contribute to the fidelity of DNA polymerases in DNA replication. The action of exonucleases in processing DNA intermediates during normal and aberrant DNA replication is then assessed, as is the importance of exonucleases in repair of double-strand breaks and interstrand crosslinks. Finally we examine how exonucleases are involved in maintenance of mitochondrial genome stability. Throughout the review, we assess how nuclease mutation or loss predisposes to a range of clinical diseases and particularly ageing.

Prospects for rejuvenation of aged tissue by telomerase reactivation.

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.

DmWRNexo is a 3'-5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease.

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.

Natural competence of mammalian mitochondria allows the molecular investigation of mitochondrial gene expression.

Respiration, a fundamental process in mammalian cells, requires two genomes, those of the nucleus and the mitochondrion (mtDNA). Mutations of mtDNA are being increasingly recognized in disease and may play an important role in the ageing process. Accepting the vital role of mtDNA gene products, our limited knowledge concerning the details of mitochondrial gene expression is surprising. This is, in part, due to our inability to transfect mitochondria and to manipulate their genome. There have been claims of successful DNA import into isolated organelles, but most reports lacked evidence of expression and no method has furthered our understanding of gene expression. Here, we report that mammalian mitochondria possess a natural competence for DNA import. Using five functional assays, we show imported DNA can act as templates for DNA synthesis or promoter-driven transcription, with the resultant polycistronic RNA being processed (5' and 3') and excised mt-tRNA matured. Exploiting this natural competence will allow us to explore mitochondrial gene expression in organello and provides the potential for mitochondrial transfection in vivo.

POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates.

Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3'-5'-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3' tail as it is exposed during unwinding.