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The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

Bailly AP, Freeman A, Hall J, Déclais AC, Alpi A, Lilley DM, Ahmed S, Gartner A - PLoS Genet. (2010)

Bottom Line: DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes.An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo.Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, United Kingdom.

ABSTRACT
DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

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Related in: MedlinePlus

Phylogenetic analysis of GEN-1 and in vitro nuclease activity.(A) Diagram showing the GEN-1 domain structure. (B) Unrooted phylogenetic tree of XPG-superfamily members. Bacterial Taq. Pol1 serves as an outgroup. Protein sequences were aligned using Jalview 2.4 employing the MAFT algorithm [83], and the Splitstree program was used to generate the tree shown using the Neighbour Joining Method. The length of the scale bar indicates the lengths of the branches of the phylogenetic trees corresponding to a 10% chance (p 0.1) of replacing an amino acid/site. An, Aspergillus nidulans, Bm, Brugia malayi, Ca, Candida albicans, Ci, Ciona intestinalis, Dm, Drosophila melanogaster, Gg, Gallus gallus, Hs, Homo sapiens, Nv, Nematostella vectensis, Pp, Pichia pasteuris, Sc, Saccharomyces cerevisiae, Sp, Schizosaccharomyces pombe. (C) Four distinct XPG-1 family members occur in all animals examined. The current view of phylogenetic relationships between vertebrates, insects and nematodes is indicated. (D) Affinity purified GEN-1 antibodies (guinea pig) detect a specific band corresponding to the predicted size of GEN-1 and GEN-1 (yp30) (for details Figure S7B, S7C). Equal loading is demonstrated by the Coomassie staining of the membrane. (E) Jbm5 (left panel) and X26 (right panel) junctions, radioactively [5′-32P]-labeled on the b and a strand respectively, were incubated at 37°C with the indicated protein (Figure S4A), and the cleavage products analyzed by denaturing gel electrophoresis in polyacrylamide. Lane 1 is a sequence marker for the b stand (left panel). The sequence of the junction is shown, with the homologous section shaded, and cleavage sites are indicated. (F) Symmetry of Holliday junction cleavage by recombinant GEN1. Holliday junction Jbm5, radioactively [5′-32P]-labeled on either the b or the d strand was incubated at 37°C with the indicated protein, and the cleavage products were analysed by electrophoresis in polyacrylamide gels under denaturing conditions. M indicates the sequence marker for each respective strand. (G) GEN-1 is specific for Holliday Junctions. Single-stranded, duplex, 3′ overhang and 5′ flap structures were subjected to GEN-1 nucleolytic activity as shown in E. For each reaction, 5 nM of gel purified substrates has been used. For more details see Materials and Methods.
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pgen-1001025-g002: Phylogenetic analysis of GEN-1 and in vitro nuclease activity.(A) Diagram showing the GEN-1 domain structure. (B) Unrooted phylogenetic tree of XPG-superfamily members. Bacterial Taq. Pol1 serves as an outgroup. Protein sequences were aligned using Jalview 2.4 employing the MAFT algorithm [83], and the Splitstree program was used to generate the tree shown using the Neighbour Joining Method. The length of the scale bar indicates the lengths of the branches of the phylogenetic trees corresponding to a 10% chance (p 0.1) of replacing an amino acid/site. An, Aspergillus nidulans, Bm, Brugia malayi, Ca, Candida albicans, Ci, Ciona intestinalis, Dm, Drosophila melanogaster, Gg, Gallus gallus, Hs, Homo sapiens, Nv, Nematostella vectensis, Pp, Pichia pasteuris, Sc, Saccharomyces cerevisiae, Sp, Schizosaccharomyces pombe. (C) Four distinct XPG-1 family members occur in all animals examined. The current view of phylogenetic relationships between vertebrates, insects and nematodes is indicated. (D) Affinity purified GEN-1 antibodies (guinea pig) detect a specific band corresponding to the predicted size of GEN-1 and GEN-1 (yp30) (for details Figure S7B, S7C). Equal loading is demonstrated by the Coomassie staining of the membrane. (E) Jbm5 (left panel) and X26 (right panel) junctions, radioactively [5′-32P]-labeled on the b and a strand respectively, were incubated at 37°C with the indicated protein (Figure S4A), and the cleavage products analyzed by denaturing gel electrophoresis in polyacrylamide. Lane 1 is a sequence marker for the b stand (left panel). The sequence of the junction is shown, with the homologous section shaded, and cleavage sites are indicated. (F) Symmetry of Holliday junction cleavage by recombinant GEN1. Holliday junction Jbm5, radioactively [5′-32P]-labeled on either the b or the d strand was incubated at 37°C with the indicated protein, and the cleavage products were analysed by electrophoresis in polyacrylamide gels under denaturing conditions. M indicates the sequence marker for each respective strand. (G) GEN-1 is specific for Holliday Junctions. Single-stranded, duplex, 3′ overhang and 5′ flap structures were subjected to GEN-1 nucleolytic activity as shown in E. For each reaction, 5 nM of gel purified substrates has been used. For more details see Materials and Methods.

Mentions: To uncover new genes involved in DNA damage response signaling, we chose an unbiased genetic approach and screened for C. elegans mutants hypersensitive to IR and/or defective in DNA damage-induced cell cycle arrest and apoptosis. During C. elegans development, the majority of cell divisions occur during embryogenesis. In contrast, germ cell proliferation, which commences with two germ cells at the L1 larval stage, predominates in the following three larval stages and continues in adult worms, where all somatic cells are post-mitotic, but continued germ cell proliferation results in a steady state level of ∼500 germ cells. In order to select for mutants hypersensitive to IR, worms mutagenised with ethyl methane sulphonate (EMS) were irradiated at the L1 stage with 60 Gy of IR. This dose of radiation does not overtly affect germ cell proliferation in wild type worms while mutants hypersensitive to IR display reduced levels of fertility (data not shown). Out of 906 F2 lines screened, 3 mutations (yp30, yp42 and yp45) were recovered for the yp30 complementation group, each of which was derived from an independently mutagenised Po animal. In C. elegans, treatment of L4 larvae with IR leads to the activation of a DNA damage response checkpoint pathway that triggers apoptosis of meiotic pachytene stage germ cells, and a transient halt of mitotic germ cell proliferation leading to enlarged cells [39]. This latter phenotype results from continued cellular growth in the absence of cell division. The yp30 complementation group does not enlarge mitotic germ cells upon IR of L4 larvae, similar to the mrt-2 (e2663) checkpoint mutant (Figure 1A and 1B, Figure S1B) [39], and is partially defective in DNA damage-induced apoptosis (Figure 1C). We did not find any further mutants, which were defective in both IR-induced cell cycle arrest and apoptosis like the yp30 complementation group (data not shown). To show that yp30 germ cells do indeed fail to arrest cell cycle progression after irradiation, we stained N2 wild type and yp30 mutants with antibodies against phosphorylated tyrosine-15 CDK-1, which serves as a G2 marker [42], [43]. We found that wild type germ cells arrest in G2, whereas yp30 germ cells fail to do so (Figure 1D), a finding we confirmed using a YFP::Cyclin B1 fusion construct as a G2 marker (Figure S1A). To clone the gene corresponding to yp30, we followed the cell cycle arrest-defective phenotype in backcrossing, SNP-mapping and complementation experiments and positioned yp30 close to the centre of chromosome III, between dpy-17 and unc-32, to an interval of approximately 135,000 base pairs (Figure 1E, data not shown). Sequencing this interval in yp30 worms revealed two mutations, one in an intergenic region, and one that leads to a premature stop codon in a gene encoding for a conserved nuclease we refer to as gen-1 (see below, Figure 1F, Figure 2A). yp42 and yp45 also contained the same C to T point mutation as gen-1(yp30), but lacked the intergenic mutation found in yp30. Sequencing of the gen-1 cDNA confirmed the predicted gen-1 cDNA sequence and the predicted intron-exon structure of gen-1, available from Wormbase (http://www.wormbase.org/; data not shown). A gen-1 deletion allele, gen-1(tm2940) (Figure 2A) obtained from the Japanese C. elegans knockout consortium, as well as gen-1 (RNAi), similarly lead to a cell cycle arrest defect upon irradiation (Figure S1B). In addition, the same phenotype was observed in gen-1(tm2940)/gen-1(yp30) trans-heterozygotes (Figure S1B). Time course and dose response experiments revealed that gen-1(tm2940), gen-1(yp30) and mrt-2(e2663) worms are equally defective in IR induced cell cycle arrest (Figure S2). Furthermore, gen-1(tm2940) is largely defective in DNA damage-induced apoptosis, similar to cep-1(lg12501), a deletion mutant of the C. elegans p53-like gene cep-1 [41], [44] (Figure 1C). In summary, our data reveal that gen-1 is required for IR-induced apoptosis and cell cycle arrest in C. elegans germ cells.


The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

Bailly AP, Freeman A, Hall J, Déclais AC, Alpi A, Lilley DM, Ahmed S, Gartner A - PLoS Genet. (2010)

Phylogenetic analysis of GEN-1 and in vitro nuclease activity.(A) Diagram showing the GEN-1 domain structure. (B) Unrooted phylogenetic tree of XPG-superfamily members. Bacterial Taq. Pol1 serves as an outgroup. Protein sequences were aligned using Jalview 2.4 employing the MAFT algorithm [83], and the Splitstree program was used to generate the tree shown using the Neighbour Joining Method. The length of the scale bar indicates the lengths of the branches of the phylogenetic trees corresponding to a 10% chance (p 0.1) of replacing an amino acid/site. An, Aspergillus nidulans, Bm, Brugia malayi, Ca, Candida albicans, Ci, Ciona intestinalis, Dm, Drosophila melanogaster, Gg, Gallus gallus, Hs, Homo sapiens, Nv, Nematostella vectensis, Pp, Pichia pasteuris, Sc, Saccharomyces cerevisiae, Sp, Schizosaccharomyces pombe. (C) Four distinct XPG-1 family members occur in all animals examined. The current view of phylogenetic relationships between vertebrates, insects and nematodes is indicated. (D) Affinity purified GEN-1 antibodies (guinea pig) detect a specific band corresponding to the predicted size of GEN-1 and GEN-1 (yp30) (for details Figure S7B, S7C). Equal loading is demonstrated by the Coomassie staining of the membrane. (E) Jbm5 (left panel) and X26 (right panel) junctions, radioactively [5′-32P]-labeled on the b and a strand respectively, were incubated at 37°C with the indicated protein (Figure S4A), and the cleavage products analyzed by denaturing gel electrophoresis in polyacrylamide. Lane 1 is a sequence marker for the b stand (left panel). The sequence of the junction is shown, with the homologous section shaded, and cleavage sites are indicated. (F) Symmetry of Holliday junction cleavage by recombinant GEN1. Holliday junction Jbm5, radioactively [5′-32P]-labeled on either the b or the d strand was incubated at 37°C with the indicated protein, and the cleavage products were analysed by electrophoresis in polyacrylamide gels under denaturing conditions. M indicates the sequence marker for each respective strand. (G) GEN-1 is specific for Holliday Junctions. Single-stranded, duplex, 3′ overhang and 5′ flap structures were subjected to GEN-1 nucleolytic activity as shown in E. For each reaction, 5 nM of gel purified substrates has been used. For more details see Materials and Methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908289&req=5

pgen-1001025-g002: Phylogenetic analysis of GEN-1 and in vitro nuclease activity.(A) Diagram showing the GEN-1 domain structure. (B) Unrooted phylogenetic tree of XPG-superfamily members. Bacterial Taq. Pol1 serves as an outgroup. Protein sequences were aligned using Jalview 2.4 employing the MAFT algorithm [83], and the Splitstree program was used to generate the tree shown using the Neighbour Joining Method. The length of the scale bar indicates the lengths of the branches of the phylogenetic trees corresponding to a 10% chance (p 0.1) of replacing an amino acid/site. An, Aspergillus nidulans, Bm, Brugia malayi, Ca, Candida albicans, Ci, Ciona intestinalis, Dm, Drosophila melanogaster, Gg, Gallus gallus, Hs, Homo sapiens, Nv, Nematostella vectensis, Pp, Pichia pasteuris, Sc, Saccharomyces cerevisiae, Sp, Schizosaccharomyces pombe. (C) Four distinct XPG-1 family members occur in all animals examined. The current view of phylogenetic relationships between vertebrates, insects and nematodes is indicated. (D) Affinity purified GEN-1 antibodies (guinea pig) detect a specific band corresponding to the predicted size of GEN-1 and GEN-1 (yp30) (for details Figure S7B, S7C). Equal loading is demonstrated by the Coomassie staining of the membrane. (E) Jbm5 (left panel) and X26 (right panel) junctions, radioactively [5′-32P]-labeled on the b and a strand respectively, were incubated at 37°C with the indicated protein (Figure S4A), and the cleavage products analyzed by denaturing gel electrophoresis in polyacrylamide. Lane 1 is a sequence marker for the b stand (left panel). The sequence of the junction is shown, with the homologous section shaded, and cleavage sites are indicated. (F) Symmetry of Holliday junction cleavage by recombinant GEN1. Holliday junction Jbm5, radioactively [5′-32P]-labeled on either the b or the d strand was incubated at 37°C with the indicated protein, and the cleavage products were analysed by electrophoresis in polyacrylamide gels under denaturing conditions. M indicates the sequence marker for each respective strand. (G) GEN-1 is specific for Holliday Junctions. Single-stranded, duplex, 3′ overhang and 5′ flap structures were subjected to GEN-1 nucleolytic activity as shown in E. For each reaction, 5 nM of gel purified substrates has been used. For more details see Materials and Methods.
Mentions: To uncover new genes involved in DNA damage response signaling, we chose an unbiased genetic approach and screened for C. elegans mutants hypersensitive to IR and/or defective in DNA damage-induced cell cycle arrest and apoptosis. During C. elegans development, the majority of cell divisions occur during embryogenesis. In contrast, germ cell proliferation, which commences with two germ cells at the L1 larval stage, predominates in the following three larval stages and continues in adult worms, where all somatic cells are post-mitotic, but continued germ cell proliferation results in a steady state level of ∼500 germ cells. In order to select for mutants hypersensitive to IR, worms mutagenised with ethyl methane sulphonate (EMS) were irradiated at the L1 stage with 60 Gy of IR. This dose of radiation does not overtly affect germ cell proliferation in wild type worms while mutants hypersensitive to IR display reduced levels of fertility (data not shown). Out of 906 F2 lines screened, 3 mutations (yp30, yp42 and yp45) were recovered for the yp30 complementation group, each of which was derived from an independently mutagenised Po animal. In C. elegans, treatment of L4 larvae with IR leads to the activation of a DNA damage response checkpoint pathway that triggers apoptosis of meiotic pachytene stage germ cells, and a transient halt of mitotic germ cell proliferation leading to enlarged cells [39]. This latter phenotype results from continued cellular growth in the absence of cell division. The yp30 complementation group does not enlarge mitotic germ cells upon IR of L4 larvae, similar to the mrt-2 (e2663) checkpoint mutant (Figure 1A and 1B, Figure S1B) [39], and is partially defective in DNA damage-induced apoptosis (Figure 1C). We did not find any further mutants, which were defective in both IR-induced cell cycle arrest and apoptosis like the yp30 complementation group (data not shown). To show that yp30 germ cells do indeed fail to arrest cell cycle progression after irradiation, we stained N2 wild type and yp30 mutants with antibodies against phosphorylated tyrosine-15 CDK-1, which serves as a G2 marker [42], [43]. We found that wild type germ cells arrest in G2, whereas yp30 germ cells fail to do so (Figure 1D), a finding we confirmed using a YFP::Cyclin B1 fusion construct as a G2 marker (Figure S1A). To clone the gene corresponding to yp30, we followed the cell cycle arrest-defective phenotype in backcrossing, SNP-mapping and complementation experiments and positioned yp30 close to the centre of chromosome III, between dpy-17 and unc-32, to an interval of approximately 135,000 base pairs (Figure 1E, data not shown). Sequencing this interval in yp30 worms revealed two mutations, one in an intergenic region, and one that leads to a premature stop codon in a gene encoding for a conserved nuclease we refer to as gen-1 (see below, Figure 1F, Figure 2A). yp42 and yp45 also contained the same C to T point mutation as gen-1(yp30), but lacked the intergenic mutation found in yp30. Sequencing of the gen-1 cDNA confirmed the predicted gen-1 cDNA sequence and the predicted intron-exon structure of gen-1, available from Wormbase (http://www.wormbase.org/; data not shown). A gen-1 deletion allele, gen-1(tm2940) (Figure 2A) obtained from the Japanese C. elegans knockout consortium, as well as gen-1 (RNAi), similarly lead to a cell cycle arrest defect upon irradiation (Figure S1B). In addition, the same phenotype was observed in gen-1(tm2940)/gen-1(yp30) trans-heterozygotes (Figure S1B). Time course and dose response experiments revealed that gen-1(tm2940), gen-1(yp30) and mrt-2(e2663) worms are equally defective in IR induced cell cycle arrest (Figure S2). Furthermore, gen-1(tm2940) is largely defective in DNA damage-induced apoptosis, similar to cep-1(lg12501), a deletion mutant of the C. elegans p53-like gene cep-1 [41], [44] (Figure 1C). In summary, our data reveal that gen-1 is required for IR-induced apoptosis and cell cycle arrest in C. elegans germ cells.

Bottom Line: DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes.An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo.Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, United Kingdom.

ABSTRACT
DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

Show MeSH
Related in: MedlinePlus