Yeast Exonuclease 5 Is Essential for Mitochondrial Genome Maintenance

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Δ strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5′ exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5′ double-stranded DNA or 5′ RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.Endonucleases and exonucleases are intimately involved in all aspects of DNA metabolism in the cell. In mitochondria, several constitutive nucleases have been identified that contribute to the proper maintenance of the mitochondrial genome through replication and recombination pathways. In addition, nucleases can localize to mitochondria in response to DNA stress in order to mediate appropriate DNA repair. Among the constitutive mitochondrial nucleases in Saccharomyces cerevisiae are the Nuc1 nuclease that contributes to DNA recombination efficiency and functions in apoptosis (4, 38) and the Cce1 endonuclease that resolves recombination intermediates (29). The Din7 endonuclease is a mitochondrially located 5′ flap endonuclease related to FEN1 (20). While deletion of the gene for either of these enzymes produced marginal mitochondrial phenotypes, more severe phenotypes were observed when combined deletions of these nuclease genes were studied or when they were combined with deletions of other genes involved in DNA recombination or repair, such as MHR1 or MSH1 (20, 22, 27). Recently, human Dna2 was shown to localize to both the nuclear and mitochondrial compartments and to participate in mitochondrial DNA replication and base excision repair (11, 39). Its function in yeast mitochondrial DNA maintenance has not been studied in detail. Finally, the 5′ flap endonuclease FEN1, which normally functions in primer RNA degradation during Okazaki fragment maturation in the nucleus, also localizes to the mitochondrion in response to DNA damage, participating in long-patch base excision repair (19, 23).Since mitochondrial function is not essential to yeast survival, dysfunction caused by mutations of the mitochondrial genome can be readily detected as a loss of respiration function, which is scored as the inability to grow on nonfermentable carbon sources. A defect in the mitochondrial DNA polymerase γ MIP1 results in complete loss of the mitochondrial DNA, and the mutant fails to grow on glycerol-containing media lacking glucose (14). Such cells are designated ρ⁰. Genome maintenance defects can also result in the generation of petite mutants that still contain mitochondrial DNA. Generally, most of the mitochondrial genome has been deleted, and a small origin-containing region has been amplified (ρ⁻). S. cerevisiae contains eight such origin regions that are highly similar in sequence and are distributed over the 86-kb mitochondrial genome (8, 9, 15). Petites that have amplified the ori5 region have been studied more extensively (16, 22).While the nucleases listed above participate in the proper maintenance of the mitochondrial genome through their replication and/or recombination functions, none appears to be essential for the integrity of the mitochondrial genome. One reasonable explanation for these observations is functional redundancy. Indeed, functional nuclease redundancy is quite common; it has been observed in the process of DNA degradation during mismatch repair in Escherichia coli, during Okazaki fragment maturation in yeast, and during the resection of double-stranded breaks in yeast (7, 25, 33). However, the possibility remains that an additional nuclease(s) is active in the mitochondrion. The present paper describes an essential mitochondrial exonuclease that is distantly related to the nuclease domain of RecB, a subunit of the bacterial RecBCD recombinase. This nuclease was discovered over 2 decades ago during a biochemical chromatographic survey of yeast exonucleases and was called exonuclease 5 (3). Initial studies with a partially purified enzyme preparation showed it to be a 5′ exonuclease specific for single-stranded DNA (ssDNA). Here we report the identification of the EXO5 gene and describe comprehensive biochemical and genetic studies that show a critical role for EXO5 in mitochondrial DNA maintenance, presumably through the processing of replication intermediates. Upon deletion of EXO5 or inactivation of its nuclease activity, only ρ⁻ mutants could be recovered. EXO5 has previously been characterized as DEM1 (defects in morphology) because the deletion mutant shows defects in growth and in mitochondrial morphology (10, 12). No nuclear defect associated with an EXO5 deletion has been detected.     

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.     

Ebselen augments its peroxidase activity by inducing nrf-2-dependent transcription

Ebselen is an organoselenium compound that acts as a glutathione peroxidase mimic. Since ebselen is a hydrophobic, thio-reactive compound capable of interacting with Keap-1, we tested its ability to activate nrf-2-dependent responses in the human hepatocarcinoma derived cell line, HepG2. Ebselen (25 microM) increased expression of an nrf-2 response element reporter in transient transfection experiments by 4-fold. Although, the induction was lower than that observed with classic nrf-2 inducer, sulforaphane (10 microL; 7-fold), ebselen also induced expression of native NAD(P)H:quinone oxidoreductase (1.6-fold) activity; induction of this protein is known to be dependent on nrf-2 action. Treatment of HepG2 cells with ebselen increased glutathione levels after 12 (1.5-fold) or 24 (1.9-fold)h of treatment. Treatment of the cells with either sulforaphane or ebselen 24 h prior to treatment with varying concentrations of t-butyl hydroperoxide increased the half maximal lethal dose from 28 to 42 microM and 58 microM for sulforaphane and ebselen, respectively. The protective effects of ebselen treatment were greater with pretreatment (IC50=58 microM) than simultaneous addition (IC50=45 microM). The protein synthesis inhibitor cycloheximide blocked increases in intracellular glutathione synthesis and partially blocked the protective effects of this regimen on increasing cell survival following t-butyl hydroperoxide treatment. Likewise co-treatment with the MEK 1 inhibitor, PD98059, which has been shown to inhibit nrf-2-dependent gene activation, partially inhibited the ebselen-dependent increases in IC50 while not affecting the control cells. We conclude that nrf-2 activation augments the role of ebselen as an antioxidant or by indirect induction of cellular antioxidant defences.     

Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.     

30-Year Trends in Stroke Rates and Outcome in Auckland,New Zealand (1981-2012): A Multi-Ethnic Population-Based Series of Studies

Background

Insufficient data exist on population-based trends in morbidity and mortality to determine the success of prevention strategies and improvements in health care delivery in stroke. The aim of this study was to determine trends in incidence and outcome (1-year mortality, 28-day case-fatality) in relation to management and risk factors for stroke in the multi-ethnic population of Auckland, New Zealand (NZ) over 30-years.

Methods

Four stroke incidence population-based register studies were undertaken in adult residents (aged ≥15 years) of Auckland NZ in 1981–1982, 1991–1992, 2002–2003 and 2011–2012. All used standard World Health Organization (WHO) diagnostic criteria and multiple overlapping sources of case-ascertainment for hospitalised and non-hospitalised, fatal and non-fatal, new stroke events. Ethnicity was consistently self-identified into four major groups. Crude and age-adjusted (WHO world population standard) annual incidence and mortality with corresponding 95% confidence intervals (CI) were calculated per 100,000 people, assuming a Poisson distribution.

Results

5400 new stroke patients were registered in four 12 month recruitment phases over the 30-year study period; 79% were NZ/European, 6% Māori, 8% Pacific people, and 7% were of Asian or other origin. Overall stroke incidence and 1-year mortality decreased by 23% (95% CI 5%-31%) and 62% (95% CI 36%-86%), respectively, from 1981 to 2012. Whilst stroke incidence and mortality declined across all groups in NZ from 1991, Māori and Pacific groups had the slowest rate of decline and continue to experience stroke at a significantly younger age (mean ages 60 and 62 years, respectively) compared with NZ/Europeans (mean age 75 years). There was also a decline in 28-day stroke case fatality (overall by 14%, 95% CI 11%-17%) across all ethnic groups from 1981 to 2012. However, there were significant increases in the frequencies of pre-morbid hypertension, myocardial infarction, and diabetes mellitus, but a reduction in frequency of current smoking among stroke patients.

Conclusions

In this unique temporal series of studies spanning 30 years, stroke incidence, early case-fatality and 1-year mortality have declined, but ethnic disparities in risk and outcome for stroke persisted suggesting that primary stroke prevention remains crucial to reducing the burden of this disease.     

Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: genotype, phenotype, ecology, worldwide spread, susceptibility, applicability

Background

Lymnaeid snails transmit medical and veterinary important trematodiases, mainly fascioliasis. Vector specificity of fasciolid parasites defines disease distribution and characteristics. Different lymnaeid species appear linked to different transmission and epidemiological patterns. Pronounced susceptibility differences to absolute resistance have been described among lymnaeid populations. When assessing disease characteristics in different endemic areas, unexpected results were obtained in studies on lymnaeid susceptibility to Fasciola. We undertook studies to understand this disease transmission heterogeneity.

Methodology/Principal Findings

A ten-year study in Iran, Egypt, Spain, the Dominican Republic, Mexico, Venezuela, Ecuador and Peru, demonstrated that such heterogeneity is not due to susceptibility differences, but to a hitherto overlooked cryptic species, Lymnaea schirazensis, confused with the main vector Galba truncatula and/or other Galba/Fossaria vectors. Nuclear rDNA and mtDNA sequences and phylogenetic reconstruction highlighted an old evolutionary divergence from other Galba/Fossaria species, and a low intraspecific variability suggesting a recent spread from one geographical source. Morphometry, anatomy and egg cluster analyses allowed for phenotypic differentiation. Selfing, egg laying, and habitat characteristics indicated a migration capacity by passive transport. Studies showed that it is not a vector species (n = 8572 field collected, 20 populations): snail finding and penetration by F. hepatica miracidium occur but never lead to cercarial production (n = 338 experimentally infected).

Conclusions/Significance

This species has been distorting fasciolid specificity/susceptibility and fascioliasis geographical distribution data. Hence, a large body of literature on G. truncatula should be revised. Its existence has henceforth to be considered in research. Genetic data on livestock, archeology and history along the 10,000-year post-domestication period explain its wide spread from the Neolithic Fertile Crescent. It is an efficient biomarker for the follow-up of livestock movements, a crucial aspect in fascioliasis emergence. It offers an outstanding laboratory model for genetic studies on susceptibility/resistance in F. hepatica/lymnaeid interaction, a field of applied research with disease control perspectives.     

Character displacement in the midst of background evolution in island populations of Anolis lizards: A spatiotemporal perspective

Negative interactions between species can generate divergent selection that causes character displacement. However, other processes cause similar divergence. We use spatial and temporal replication across island populations of Anolis lizards to assess the importance of negative interactions in driving trait shifts. Previous work showed that the establishment of Anolis sagrei on islands drove resident Anolis carolinensis to perch higher and evolve larger toepads. To further test the interaction's causality and predictability, we resurveyed a subset of islands nine years later. Anolis sagrei had established on one island between surveys. We found that A. carolinensis on this island now perch higher and have larger toepads. However, toepad morphology change on this island was not distinct from shifts on six other islands whose Anolis community composition had not changed. Thus, the presence of A. sagrei only partly explains A. carolinensis trait variation across space and time. We also found that A. carolinensis on islands with previously established A. sagrei now perch higher than a decade ago, and that current A. carolinensis perch height is correlated with A. sagrei density. Our results suggest that character displacement likely interacts with other evolutionary processes in this system, and that temporal data are key to detecting such interactions.