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Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair, somatic and meiotic recombination.

Bonnet S, Knoll A, Hartung F, Puchta H - Nucleic Acids Res. (2013)

Bottom Line: Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants.AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains.To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains.

View Article: PubMed Central - PubMed

Affiliation: Karlsruhe Institute of Technology, Botanical Institute II, Hertzstrasse 16, 76187 Karlsruhe, Germany.

ABSTRACT
Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.

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Complementation of the cisplatin and MMS sensitivity of Atrmi1-2. In all depicted experiments, a final cisplatin concentration of 10 µM and a final MMS concentration of 60 ppm was used. (A) The expression of the wild-type RMI1 construct enables the complementation of the hypersensitivity of Atrmi1-2 against cisplatin. (B and C) The constructs RMI1ΔDUF and RMI1ΔOB1 cannot compensate for the elevated sensitivity against cisplatin. (D) In comparison with RMI1ΔDUF and RMI1ΔOB1, with the recombinant protein RMI1ΔOB2, some lines show complementation of the hypersensitivity against cisplatin. (E) Expression of wild-type RMI1 in Atrmi1-2 rescues the hypersensitivity of the mutant against MMS. (F and G) Lines expressing the constructs RMI1ΔDUF and RMI1ΔOB1 cannot repair MMS-induced DNA damage better than the Atrmi1-2 mutant line. (H) Most lines expressing a construct of RMI1 missing the OB2 domain display a repair capacity of MMS-induced DNA damage that is higher than that of the Atrmi1-2 mutant. All experiments n = 3.
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gkt730-F3: Complementation of the cisplatin and MMS sensitivity of Atrmi1-2. In all depicted experiments, a final cisplatin concentration of 10 µM and a final MMS concentration of 60 ppm was used. (A) The expression of the wild-type RMI1 construct enables the complementation of the hypersensitivity of Atrmi1-2 against cisplatin. (B and C) The constructs RMI1ΔDUF and RMI1ΔOB1 cannot compensate for the elevated sensitivity against cisplatin. (D) In comparison with RMI1ΔDUF and RMI1ΔOB1, with the recombinant protein RMI1ΔOB2, some lines show complementation of the hypersensitivity against cisplatin. (E) Expression of wild-type RMI1 in Atrmi1-2 rescues the hypersensitivity of the mutant against MMS. (F and G) Lines expressing the constructs RMI1ΔDUF and RMI1ΔOB1 cannot repair MMS-induced DNA damage better than the Atrmi1-2 mutant line. (H) Most lines expressing a construct of RMI1 missing the OB2 domain display a repair capacity of MMS-induced DNA damage that is higher than that of the Atrmi1-2 mutant. All experiments n = 3.

Mentions: The enhanced sensitivity of Atrmi1-2, Atrecq4A and Attop3a against cisplatin and MMS was previously shown by Hartung et al. (23,24). The complementation of the hypersensitive Atrmi1-2 mutant phenotype against cisplatin and MMS was analysed by the determination of the fresh weight of the plants challenged with either the cross-linking agent cisplatin (10 µM) or the alkylating agent MMS (60 ppm) in comparison with untreated plants. After 1 week of growing on solid medium, the plants were transferred to liquid medium and treated with cisplatin or MMS for two additional weeks. The complementation of the enhanced cisplatin and MMS sensitivity of the Atrmi1-2 mutant was achieved by the expression of the wild-type AtRMI1 construct (Figure 3A and E). Lines 2, 3 and 4 could fully complement the Atrmi1-2 sensitivity against cisplatin and MMS. Line #1 showed only a partial complementation, but a significant decrease in cisplatin sensitivity (P < 0.001, Student’s t-test) and MMS sensitivity (P = 0.005, Student’s t-test) compared with the mutant line. The recombinant proteins RMI1ΔDUF and RMI1ΔOB1 could neither complement the enhanced sensitivity against cisplatin nor the enhanced sensitivity against MMS. For both constructs, the four tested lines are similar in their sensitivity to the Atrmi1-2 mutant line (Figure 3B, C, F and G). In contrast to the constructs lacking the DUF1767 domain or the OB-fold domain 1, in lines expressing the construct RMI1ΔOB2, some could fully complement the hypersensitivity of the mutant line against both cisplatin and MMS: Lines 14 and 15 showed a sensitivity almost at wild-type level, whereas lines 13 and 16 showed hardly any complementation (Figure 3D and G).Figure 3.


Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair, somatic and meiotic recombination.

Bonnet S, Knoll A, Hartung F, Puchta H - Nucleic Acids Res. (2013)

Complementation of the cisplatin and MMS sensitivity of Atrmi1-2. In all depicted experiments, a final cisplatin concentration of 10 µM and a final MMS concentration of 60 ppm was used. (A) The expression of the wild-type RMI1 construct enables the complementation of the hypersensitivity of Atrmi1-2 against cisplatin. (B and C) The constructs RMI1ΔDUF and RMI1ΔOB1 cannot compensate for the elevated sensitivity against cisplatin. (D) In comparison with RMI1ΔDUF and RMI1ΔOB1, with the recombinant protein RMI1ΔOB2, some lines show complementation of the hypersensitivity against cisplatin. (E) Expression of wild-type RMI1 in Atrmi1-2 rescues the hypersensitivity of the mutant against MMS. (F and G) Lines expressing the constructs RMI1ΔDUF and RMI1ΔOB1 cannot repair MMS-induced DNA damage better than the Atrmi1-2 mutant line. (H) Most lines expressing a construct of RMI1 missing the OB2 domain display a repair capacity of MMS-induced DNA damage that is higher than that of the Atrmi1-2 mutant. All experiments n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814364&req=5

gkt730-F3: Complementation of the cisplatin and MMS sensitivity of Atrmi1-2. In all depicted experiments, a final cisplatin concentration of 10 µM and a final MMS concentration of 60 ppm was used. (A) The expression of the wild-type RMI1 construct enables the complementation of the hypersensitivity of Atrmi1-2 against cisplatin. (B and C) The constructs RMI1ΔDUF and RMI1ΔOB1 cannot compensate for the elevated sensitivity against cisplatin. (D) In comparison with RMI1ΔDUF and RMI1ΔOB1, with the recombinant protein RMI1ΔOB2, some lines show complementation of the hypersensitivity against cisplatin. (E) Expression of wild-type RMI1 in Atrmi1-2 rescues the hypersensitivity of the mutant against MMS. (F and G) Lines expressing the constructs RMI1ΔDUF and RMI1ΔOB1 cannot repair MMS-induced DNA damage better than the Atrmi1-2 mutant line. (H) Most lines expressing a construct of RMI1 missing the OB2 domain display a repair capacity of MMS-induced DNA damage that is higher than that of the Atrmi1-2 mutant. All experiments n = 3.
Mentions: The enhanced sensitivity of Atrmi1-2, Atrecq4A and Attop3a against cisplatin and MMS was previously shown by Hartung et al. (23,24). The complementation of the hypersensitive Atrmi1-2 mutant phenotype against cisplatin and MMS was analysed by the determination of the fresh weight of the plants challenged with either the cross-linking agent cisplatin (10 µM) or the alkylating agent MMS (60 ppm) in comparison with untreated plants. After 1 week of growing on solid medium, the plants were transferred to liquid medium and treated with cisplatin or MMS for two additional weeks. The complementation of the enhanced cisplatin and MMS sensitivity of the Atrmi1-2 mutant was achieved by the expression of the wild-type AtRMI1 construct (Figure 3A and E). Lines 2, 3 and 4 could fully complement the Atrmi1-2 sensitivity against cisplatin and MMS. Line #1 showed only a partial complementation, but a significant decrease in cisplatin sensitivity (P < 0.001, Student’s t-test) and MMS sensitivity (P = 0.005, Student’s t-test) compared with the mutant line. The recombinant proteins RMI1ΔDUF and RMI1ΔOB1 could neither complement the enhanced sensitivity against cisplatin nor the enhanced sensitivity against MMS. For both constructs, the four tested lines are similar in their sensitivity to the Atrmi1-2 mutant line (Figure 3B, C, F and G). In contrast to the constructs lacking the DUF1767 domain or the OB-fold domain 1, in lines expressing the construct RMI1ΔOB2, some could fully complement the hypersensitivity of the mutant line against both cisplatin and MMS: Lines 14 and 15 showed a sensitivity almost at wild-type level, whereas lines 13 and 16 showed hardly any complementation (Figure 3D and G).Figure 3.

Bottom Line: Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants.AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains.To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains.

View Article: PubMed Central - PubMed

Affiliation: Karlsruhe Institute of Technology, Botanical Institute II, Hertzstrasse 16, 76187 Karlsruhe, Germany.

ABSTRACT
Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.

Show MeSH
Related in: MedlinePlus