Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining genome stability, morphogenesis, and life span

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.     

VIG-1 is required for maintenance of genome stability in Caenorhabditis elegans

To explore the function of VIG-1 in Caenorhabditis elegans, we analyzed the phenotypes of two vig-1 deletion mutants: vig-1(tm3383) and vig-1(ok2536). Both vig-1 mutants exhibited phenotypes associated with genome instability, such as a high incidence of males (Him) and increased embryonic lethality. These phenotypes became more evident in succeeding generations, implying that the germline of vig-1 accumulates DNA damage over generations. To examine whether vig-1 causes a defect in the DNA damage response, we treated worms with UV or camptothecin, a specific topoisomerase I inhibitor. We observed that the embryonic survival of the vig-1 mutants was reduced compared with that of the wild-type worms. Our results thus suggest that VIG-1 is required for maintaining genome stability in response to endogenous and exogenous genotoxic stresses.     

Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. RESULTS: We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. CONCLUSIONS: BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.     

The lysosomal cathepsin protease CPL-1 plays a leading role in phagosomal degradation of apoptotic cells in Caenorhabditis elegans

During programmed cell death, the clearance of apoptotic cells is achieved by their phagocytosis and delivery to lysosomes for destruction in engulfing cells. However, the role of lysosomal proteases in cell corpse destruction is not understood. Here we report the identification of the lysosomal cathepsin CPL-1 as an indispensable protease for apoptotic cell removal in Caenorhabditis elegans. We find that loss of cpl-1 function leads to strong accumulation of germ cell corpses, which results from a failure in degradation rather than engulfment. CPL-1 is expressed in a variety of cell types, including engulfment cells, and its mutation does not affect the maturation of cell corpse–containing phagosomes, including phagosomal recruitment of maturation effectors and phagosome acidification. Of importance, we find that phagosomal recruitment and incorporation of CPL-1 occurs before digestion of cell corpses, which depends on factors required for phagolysosome formation. Using RNA interference, we further examine the role of other candidate lysosomal proteases in cell corpse clearance but find that they do not obviously affect this process. Collectively, these findings establish CPL-1 as the leading lysosomal protease required for elimination of apoptotic cells in C. elegans.     

Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis elegans

In C. elegans, DOG-1 prevents deletions that initiate in polyG/polyC tracts (G/C tracts), most likely by unwinding secondary structures that can form in G/C tracts during lagging-strand DNA synthesis. We have used the dog-1 mutant to assay the in vivo contribution of various repair genes to the maintenance of G/C tracts. Here we show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G/C tracts. Similarly, we demonstrate that the C. elegans orthologs of the homologous recombination repair genes BARD1, RAD51, and XPF and the trans-lesion synthesis polymerases poleta and polkappa contribute to the prevention of deletions in dog-1 mutants. Finally, we provide evidence that the small deletions generated in the dog-1 background are not formed through homologous recombination, nucleotide excision repair, or nonhomologous end-joining mechanisms, but appear to result from a mutagenic repair mechanism acting at G/C tracts. Our data support the hypothesis that absence of DOG-1 leads to replication fork stalling that can be repaired by deletion-free or deletion-prone mechanisms.     

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Serpins in the Caenorhabditis elegans genome.

Data mining in genome sequences can identify distant homologues of known protein families, and is most powerful if solved structures are available to reveal the three-dimensional implications of very dissimilar sequences. Here we describe putative serpin sequences identified with very high statistical significance in the Caenorhabditis elegans genome. When mapped onto vertebrate serpins such as alpha1-antitrypsin, they suggest novel structural features. Some appear complete, some show extensive deletions, and others appear to contain only the C-terminal part of the known serpin fold, probably in partnership with N-terminal regions that have conformations unlike those of known serpins. The observation of such striking sequence similarity, in proteins that must have significantly different overall structures, substantially extends the structural characteristics of the serpin family of proteins.     

线虫(Caenorhabditis elegans)基因组的研究进展

线虫（Ｃａｅｎｏｒｈａｂｄｉｔｉｓ ｅｌｅｇａｎｓ）是重要的模式生物,其基因组序列分析工作于１９９８年底基本完成,已有１９０００多个基因被鉴定。本文概述线虫基因组研究中遗传图谱、物理图谱、序列测定和基因识别等方面的研究成果,以及线虫基因组计划将对生命科学研究产生的影响。     

Minichromosome maintenance protein 5 homologue in Caenorhabditis elegans plays essential role for postembryonic development

Genome duplication is tightly controlled in multicellular organisms to ensure the genome stability. Studies in Saccharomyces cerevisiae and Xenopus show that minichromosome maintenance (MCM) proteins are essential for genome duplication. However, the development role of MCM proteins in multicellular organisms is not well known. MCM5 encodes a member of the MCM2-7 protein family involved in the initiation of DNA replication. The sequences of all Mcm5 homologues from yeast to human are highly conserved and suggest that their functions are also conserved. Here, we isolated the first mutant allele of mcm-5 (fw7) in Caenorhabditis elegans. Homozygous mcm-5 (fw7) mutants from heterozygous parents exhibited variable larval lethality and adult sterility. The postembryonically born neuron number was decreased and also showed aberrant axon morphology. Our study revealed that the losses of neurons in mcm-5 (fw7) mutants were caused by cell cycle defects not by programmed cell death. The examination showed that mcm-5 was widely used for postembryonic development in multiple cells such as seam cells, gonad and intestinal cells. Knockdown of mcm-5 by RNAi caused 98.1% embryonic arrest, suggesting that mcm-5 was also required for embryonic development. After RNAi treatment of the other MCM2-7 family members, we found that they all exhibited similar phenotypes as mcm-5, suggesting that the MCM2-7 family in C. elegans might function associated with cell division as its homologues in S. cerevisiae.     

CRISPR-Cas9基因编辑技术在秀丽线虫中的应用

基于细菌基因组规律成蔟的间隔短回文重复(Clustered regularly interspaced short palindromic repeats)发展而来的新型基因编辑方法(CRISPR-Cas9)对生物医学研究是一场划时代的革命。它几乎可用于大多数生物体的基因编辑。秀丽线虫是一种非常经典的遗传学模式生物,CRISPR-Cas9基因编辑技术进一步加速了对其基因功能及各种生物学问题的研究。文中主要总结CRISPR-Cas9基因编辑系统在遗传学模式生物秀丽线虫中的发展和应用。     

CemaT1 is an active transposon within the Caenorhabditis elegans genome

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. In the nematode Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in C. elegans and that the CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of C. elegans is comparable to that of the Tc1 transposon. However, unlike Tc transposons in mutator strains of C. elegans, maT transposons do not exhibit increased frequencies of mobility, suggesting that maT is not regulated by the same factors that control Tc activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the C. elegans genome.     

RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens

Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear‐encoded chloroplast‐localized homolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P. patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13–63 bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.     

The Caenorhabditis elegans homolog of the putative prostate cancer susceptibility gene ELAC2, hoe-1, plays a role in germline proliferation

The potential prostate cancer susceptibility gene ELAC2 has a Caenorhabditis elegans homolog (which we call hoe-1, for homolog of ELAC2). We have explored the biological role of this gene using RNAi to reduce gene activity. We found that worms subjected to hoe-1 RNAi are slow-growing and sterile. The sterility results from a drastic reduction in germline proliferation and cell-cycle arrest of germline nuclei. We found that hoe-1 is required for hyperproliferation phenotypes seen with mutations in three different genes, suggesting hoe-1 may be generally required for germline proliferation. We also found that reduction of hoe-1 by RNAi suppresses the multivulva (Muv) phenotype resulting from activating mutations in ras and that this suppression is likely to be indirect. This is the first demonstration of a biological role for this class of proteins in a complex eukaryote and adds important information when considering the role of ELAC2 in prostate cancer.     

piRNAs and siRNAs collaborate in Caenorhabditis elegans genome defense

ABSTRACT: Caenorhabditis elegans piRNAs promote genome surveillance by triggering siRNA-mediated silencing of nonself DNA in competition with licensing programs that support endogenous gene expression.     

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3^fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3^−/− cells. Notably, Ada3^−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3^fl/fl and Ada3^−/− cells confirmed higher residual DNA damage in Ada3^−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability.     

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3^fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3^?/? cells. Notably, Ada3^?/? cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G? or G? phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3^fl/fl and Ada3^?/? cells confirmed higher residual DNA damage in Ada3^?/? cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability.     

Hypothesis: Iron chelation plays a vital role in neutrophilic inflammation

Neutrophil influx into tissues occurs in many diverse diseases and can be associated with both beneficial and injurious effects. We hypothesize that the stimulus for certain neutrophilic inflammatory responses can be reduced to a series of competing reactions for iron, with either a labile or reactive coordination site available, between host chelators and chelators not indigenous to that specific living system. The iron focuses the transport of host phagocytic cells through a metal catalyzed generation of oxidant sensitive mediators including cytokines and eicosanoids. Many of these products are chemotactic for neutrophils. We also postulate that the iron increases the activity of the phagocyte associated NADPH oxidoreductase in the neutrophil. The function of this enzyme is likely to be the generation of superoxide in the hostÕs attempt to chemically reduce and dislodge the iron from its chelate complex. After the reoxidation of Fe in an aerobic environment, Fe will be coordinated by host lactoferrin released by the neutrophil. When complexed by this glycoprotein, the metal does not readily undergo oxidation/reduction and is safely transported to the macrophages of the reticuloendothelial system where it is stored in ferritin. Finally, we propose that the neutrophil will attempt to destroy the chelator not indigenous to the host by releasing granular contents other than lactoferrin. Inability to eliminate the chelator allows this sequence to repeat itself, which can lead to tissue injury. Such persistence of a metal chelate in the host may be associated with biomineralization, fibrosis, and cancer.