Ivan Boubriak

UV irradiation induces homologous recombination genes in the model archaeon, Halobacterium sp. NRC-1.

BackgroundA variety of strategies for survival of UV irradiation are used by cells, ranging from repair of UV-damaged DNA, cell cycle arrest, tolerance of unrepaired UV photoproducts, and shielding from UV light. Some of these responses involve UV-inducible genes, including the SOS response in bacteria and an array of genes in eukaryotes. To address the mechanisms used in the third branch of life, we have studied the model archaeon, Halobacterium sp. strain NRC-1, which tolerates high levels of solar radiation in its natural hypersaline environment.ResultsCells were irradiated with 30–70 J/m2 UV-C and an immunoassay showed that the resulting DNA damage was largely repaired within 3 hours in the dark. Under such conditions, transcriptional profiling showed the most strongly up-regulated gene was radA1, the archaeal homolog of rad51/recA, which was induced 7-fold. Additional genes involved in homologous recombination, such as arj1 (recJ-like exonuclease), dbp (eukaryote-like DNA binding protein of the superfamily I DNA and RNA helicases), and rfa3 (replication protein A complex), as well as nrdJ, encoding for cobalamin-dependent ribonucleotide reductase involved in DNA metabolism, were also significantly induced in one or more of our experimental conditions. Neither prokaryotic nor eukaryotic excision repair gene homologs were induced and there was no evidence of an SOS-like response.ConclusionThese results show that homologous recombination plays an important role in the cellular response of Halobacterium sp. NRC-1 to UV damage. Homologous recombination may permit rescue of stalled replication forks, and/or facilitate recombinational repair. In either case, this provides a mechanism for the observed high-frequency recombination among natural populations of halophilic archaea.

Mutations in the gene for the E1β subunit: a novel cause of pyruvate dehydrogenase deficiency

We describe two unrelated patients with pyruvate dehydrogenase (PDH) deficiency attributable to mutations in the gene encoding the E1β subunit of the complex. This is a previously unrecognised form of PDH deficiency, which most commonly results from mutations in the X-linked gene for the E1α subunit. Both patients had reduced immunoreactive E1β protein and both had missense mutations in the E1β gene. Activity of the PDH complex was restored in cultured fibroblasts from both patients by transfection and expression of the normal E1β coding sequence.

Transcriptional responses to biologically relevant doses of UV-B radiation in the model archaeon, Halobacterium sp. NRC-1.

BackgroundMost studies of the transcriptional response to UV radiation in living cells have used UV doses that are much higher than those encountered in the natural environment, and most focus on short-wave UV (UV-C) at 254 nm, a wavelength that never reaches the Earths surface. We have studied the transcriptional response of the sunlight-tolerant model archaeon, Halobacterium sp. NRC-1, to low doses of mid-wave UV (UV-B) to assess its response to UV radiation that is likely to be more biologically relevant.ResultsHalobacterium NRC-1 cells were irradiated with UV-B at doses equivalent to 30 J/m2 and 5 J/m2 of UV-C. Transcriptional profiling showed that only 11 genes were up-regulated 1.5-fold or more by both UV-B doses. The most strongly up-regulated gene was radA1 (vng2473), the archaeal homologue of RAD51/recA recombinase. The others included arj1 (vng779) (recJ-like exonuclease), top6A (vng884) and top6B (vng885) (coding for Topoisomerase VI subunits), and nrdJ (vng1644) (which encodes a subunit of ribonucleotide reductase). We have found that four of the consistently UV-B up-regulated genes, radA1 (vng2473), vng17, top6B (vng885) and vng280, share a common 11-base pair motif in their promoter region, TTTCACTTTCA. Similar sequences were found in radA promoters in other halophilic archaea, as well as in the radA promoter of Methanospirillum hungatei. We analysed the transcriptional response of a repair-deficient ΔuvrA (vng2636) ΔuvrC (vng2381) double-deletion mutant and found common themes between it and the response in repair proficient cells.ConclusionOur results show a core set of genes is consistently up-regulated after exposure to UV-B light at low, biologically relevant doses. Eleven genes were up-regulated, in wild-type cells, after two UV-B doses (comparable to UV-C doses of 30 J/m2 and 5 J/m2), and only four genes were up-regulated by all doses of UV-B and UV-C that we have used in this work and previously. These results suggest that high doses of UV-C radiation do not necessarily provide a good model for the natural response to environmental UV. We have found an 11-base pair motif upstream of the TATA box in four of the UV-B up-regulated genes and suggest that this motif is the binding site for a transcriptional regulator involved in their response to UV damage in this model archaeon.

Desiccation and survival in the recalcitrant seeds of Avicennia marina: DNA replication, DNA repair and protein synthesis

An autoradiographic study was made of leucine and thymidine incorporation into the meristematic root primordia and hypocotyl tips of seeds of the recalcitrant mangrove species, Avicennia marina. The investigations show that although there is a temporary reduction of protein synthesis at shedding, root primordia and surrounding hypocotyl cells of the axis never wholly cease incorporation of [ 3 H]leucine and regain preshedding levels of activity within a day. Precursor studies using methyl-[ 3 H]thymidine show that, at shedding, there is a temporary cessation of incorporation into root meristem nuclei that lasts no longer than 48 h and, within a day, pre-shedding levels are regained in the meristem nuclei. Analysis of DNA fragmentation patterns in root tips at the time of shedding, and their ability to repair radiation-induced DNA damage, indicate that DNA repair processes are markedly compromised in these cells if water loss reaches 22%. Protein synthesis and DNA replication are reduced by more than half by a water loss of 18% and 16%, respectively. DNA replication does not fully recover on rehydration after only 8% water loss. DNA fragmentation to nucleosomes indicates a programme of cell death at a water loss of 10%. We suggest that the feature of continuous protein synthesis activity with only a temporary interruption in active cell cycling in A. marina root primordia helps to explain both the rapidity in seedling establishment and the extreme vulnerability to desiccation.

Identification and characterization of a Drosophila ortholog of WRN exonuclease that is required to maintain genome integrity.

The premature human aging Werner syndrome (WS) is caused by mutation of the RecQ‐family WRN helicase, which is unique in possessing also 3′–5′ exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5′ UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hypomorphic; although adults show no gross morphological abnormalities, females are sterile. Like human WS cells, we show that the DmWRNexo mutant flies are hypersensitive to the topoisomerase I inhibitor camptothecin. Furthermore, these mutant flies show highly elevated rates of mitotic DNA recombination resulting from excessive reciprocal exchange. This study identifies a novel WRN ortholog in flies and demonstrates an important role for WRN exonuclease in maintaining genome stability.

The Achene Mucilage Hydrated in Desert Dew Assists Seed Cells in Maintaining DNA Integrity: Adaptive Strategy of Desert Plant Artemisia sphaerocephala

Despite proposed ecological importance of mucilage in seed dispersal, germination and seedling establishment, little is known about the role of mucilage in seed pre-germination processes. Here we investigated the role of mucilage in assisting achene cells to repair DNA damage during dew deposition in the desert. Artemisia sphaerocephala achenes were first treated γ-irradiation to induce DNA damage, and then they were repaired in situ in the desert dew. Dew deposition duration can be as long as 421 min in early mornings. Intact achenes absorbed more water than demucilaged achenes during dew deposition and also carried water for longer time following sunrise. After 4-d dew treatment, DNA damage of irradiated intact and demucilaged achenes was reduced to 24.38% and 46.84%, respectively. The irradiated intact achenes exhibited much higher DNA repair ratio than irradiated demucilaged achenes. Irradiated intact achenes showed an improved germination and decreased nonviable achenes after dew treatment, and significant differences in viability between the two types of achenes were detected after 1020 min of dew treatment. Achene mucilage presumably plays an ecologically important role in the life cycle of A. sphaerocephala by aiding DNA repair of achene cells in genomic-stressful habitats.

Lysine Acetylation Controls Local Protein Conformation by Influencing Proline Isomerization

Summary Gene transcription responds to stress and metabolic signals to optimize growth and survival. Histone H3 (H3) lysine 4 trimethylation (K4me3) facilitates state changes, but how levels are coordinated with the environment is unclear. Here, we show that isomerization of H3 at the alanine 15-proline 16 (A15-P16) peptide bond is influenced by lysine 14 (K14) and controls gene-specific K4me3 by balancing the actions of Jhd2, the K4me3 demethylase, and Spp1, a subunit of the Set1 K4 methyltransferase complex. Acetylation at K14 favors the A15-P16trans conformation and reduces K4me3. Environmental stress-induced genes are most sensitive to the changes at K14 influencing H3 tail conformation and K4me3. By contrast, ribosomal protein genes maintain K4me3, required for their repression during stress, independently of Spp1, K14, and P16. Thus, the plasticity in control of K4me3, via signaling to K14 and isomerization at P16, informs distinct gene regulatory mechanisms and processes involving K4me3.

Modeling Werner Syndrome in Drosophila melanogaster: hyper-recombination in flies lacking WRN-like exonuclease.

Abstract:  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.

Long term effects of Chernobyl contamination on DNA repair function and plant resistance to different biotic and abiotic stress factors

Thirty years after the Chernobyl explosion we still lack information regarding the genetic effects of radionuclide contamination on the plant population. For example, are plants adapting to the low dose of chronic ionising irradiation and showing improved resistance to radiation damage? Are they coping with changing/increased pathogenicity of fungi and viruses in the Chernobyl exclusion (ChE) zone? Are plant populations rapidly accumulating mutational load and should we expect rapid micro-evolutionary changes in plants in the Chernobyl area? This review will try to summarise the current knowledge on these aspects of plant genetics and ecology and draw conclusions on the importance of further studies in the area around Chernobyl.

The Drosophila orthologue of progeroid human WRN exonuclease, DmWRNexo, cleaves replication substrates but is inhibited by uracil or abasic sites

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 Mg2+, 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.