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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

Persistent RAD-51 foci in gen-1(tm2940) and gen-1(yp30) worms.Rad-51 foci were scored 48 h post IR with 30 Gy as described [82]. A layer of only five z-stacks is displayed for clarity. DAPI and RAD-51 correspond to blue and red staining respectively. A quantification of foci per mitotic germ line is shown in the right panel. n = 5, error bars represent s.e.m. Foci were counted by projecting all the z-stacks using SoftWorks. Scale bar is 10 µm.
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pgen-1001025-g004: Persistent RAD-51 foci in gen-1(tm2940) and gen-1(yp30) worms.Rad-51 foci were scored 48 h post IR with 30 Gy as described [82]. A layer of only five z-stacks is displayed for clarity. DAPI and RAD-51 correspond to blue and red staining respectively. A quantification of foci per mitotic germ line is shown in the right panel. n = 5, error bars represent s.e.m. Foci were counted by projecting all the z-stacks using SoftWorks. Scale bar is 10 µm.

Mentions: To test if the IR sensitivity phenotypes of gen-1 mutants correlate with persistence of DSBs, we assayed for RAD-51 foci. At doses where multiple DSBs per cell are generated, the number of persistent RAD-51 foci in mitotic germ cells of mrt-2(e2663) and both gen-1 mutants is higher as compared to wild type, indicating a DSB repair defect (Figure 4). To directly confirm whether IR leads to increased DNA double-strand breakage in gen-1 mutant worms we directly assayed for chromosome fragmentation after irradiation with 90 Gy. As shown previously [59], 48 hours after irradiation of mitotic germ cells (at the L4 stage) the diakinesis chromosomes of resulting mrt-2(e2663) oocytes were fragmented. In contrast, IR-induced damage was repaired in wild type, where oocyte chromosomes appear as 6 morphologically intact condensed DAPI stained structures (Figure 5A and 5B). Chromosome fragmentation for both gen-1 mutants was as strong as that observed for the mrt-2 positive control, indicating a defect in DSB repair. This chromosome fragmentation phenotype was not observed as a consequence of irradiating pachytene stage cells and observing corresponding oocytes ∼8 hours and ∼20 hours after IR (Figure 5C). Given that gen-1(tm2940) and gen-1(yp30) are equally defective in repairing diakinesis chromosomes 48 hours after irradiation we consider it likely that the checkpoint functions of gen-1 (and mrt-2) in mitotic germ cells contribute to DSB repair. Given that DSBs inflicted in pachytene cells are repaired in gen-1 and mrt-2 mutants while this is not the case for DSBs in mitotic germ cells there might be a stronger requirement of GEN-1 and MRT-2 for DSB repair in mitotic 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)

Persistent RAD-51 foci in gen-1(tm2940) and gen-1(yp30) worms.Rad-51 foci were scored 48 h post IR with 30 Gy as described [82]. A layer of only five z-stacks is displayed for clarity. DAPI and RAD-51 correspond to blue and red staining respectively. A quantification of foci per mitotic germ line is shown in the right panel. n = 5, error bars represent s.e.m. Foci were counted by projecting all the z-stacks using SoftWorks. Scale bar is 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908289&req=5

pgen-1001025-g004: Persistent RAD-51 foci in gen-1(tm2940) and gen-1(yp30) worms.Rad-51 foci were scored 48 h post IR with 30 Gy as described [82]. A layer of only five z-stacks is displayed for clarity. DAPI and RAD-51 correspond to blue and red staining respectively. A quantification of foci per mitotic germ line is shown in the right panel. n = 5, error bars represent s.e.m. Foci were counted by projecting all the z-stacks using SoftWorks. Scale bar is 10 µm.
Mentions: To test if the IR sensitivity phenotypes of gen-1 mutants correlate with persistence of DSBs, we assayed for RAD-51 foci. At doses where multiple DSBs per cell are generated, the number of persistent RAD-51 foci in mitotic germ cells of mrt-2(e2663) and both gen-1 mutants is higher as compared to wild type, indicating a DSB repair defect (Figure 4). To directly confirm whether IR leads to increased DNA double-strand breakage in gen-1 mutant worms we directly assayed for chromosome fragmentation after irradiation with 90 Gy. As shown previously [59], 48 hours after irradiation of mitotic germ cells (at the L4 stage) the diakinesis chromosomes of resulting mrt-2(e2663) oocytes were fragmented. In contrast, IR-induced damage was repaired in wild type, where oocyte chromosomes appear as 6 morphologically intact condensed DAPI stained structures (Figure 5A and 5B). Chromosome fragmentation for both gen-1 mutants was as strong as that observed for the mrt-2 positive control, indicating a defect in DSB repair. This chromosome fragmentation phenotype was not observed as a consequence of irradiating pachytene stage cells and observing corresponding oocytes ∼8 hours and ∼20 hours after IR (Figure 5C). Given that gen-1(tm2940) and gen-1(yp30) are equally defective in repairing diakinesis chromosomes 48 hours after irradiation we consider it likely that the checkpoint functions of gen-1 (and mrt-2) in mitotic germ cells contribute to DSB repair. Given that DSBs inflicted in pachytene cells are repaired in gen-1 and mrt-2 mutants while this is not the case for DSBs in mitotic germ cells there might be a stronger requirement of GEN-1 and MRT-2 for DSB repair in mitotic 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