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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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Both N-terminal domains DUF1767 and OB1 have different roles in suppressing HR. The recombination frequencies of (A) untreated and (B) cisplatin (3 µM) treated plants. In contrast to the RMI1ΔDUF and RMI1ΔOB1 constructs, the expression of the recombinant RMI1ΔDUFΔOB1 construct does not lead to any type of complementation of the hyper-recombination phenotype of the Atrmi1-2 mutant. All experiments n = 3.
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gkt730-F5: Both N-terminal domains DUF1767 and OB1 have different roles in suppressing HR. The recombination frequencies of (A) untreated and (B) cisplatin (3 µM) treated plants. In contrast to the RMI1ΔDUF and RMI1ΔOB1 constructs, the expression of the recombinant RMI1ΔDUFΔOB1 construct does not lead to any type of complementation of the hyper-recombination phenotype of the Atrmi1-2 mutant. All experiments n = 3.

Mentions: The previous results indicated that both N-terminal domains DUF1767 and OB1 have some function in the suppression of somatic HR. However, both could be involved in the same type of reaction that would require AtRMI1 for some but not all forms of HR suppression detected by our assay system. On the other hand, both domains might contribute, at least partially, in an independent way to the suppression phenotype so that the loss of both domains would lead to a complete loss of suppression. To discriminate between these possibilities, we generated a construct missing both the DUF1767 domain and the OB-fold domain 1 (Figure 2). After transformation of the Atrmi1-2 mutant, four different independent transgenic lines were isolated and analysed. The expression of the recombinant protein RMI1ΔDUFΔOB1 did not significantly change the frequency of HR of the Atrmi1-2 mutant either with or without cisplatin treatment (Figure 5). On the other hand, the HR frequency of all lines was significantly different from that of the wild-type without (#17: P = 0.03; #18: P = 0.02; #19: P = 0.003; #20: P < 0.001, Student’s t-test) or with cisplatin treatment (#17: P = 0.001; #18: P = 0.003; #19: P = 0.004; #20: P = 0.01, Student’s t-test). The fact that Atrmi1 plants expressing an AtRMI1 protein without both the DUF1767 and OB domain 1 show a recombination frequency that is higher than that of plants expressing an AtRMI1 where only one of the domains is missing (compare Figure 5 with Figure 3B and C; Supplementary Figure S3B and C) indicates that both domains have at least partially non-overlapping functions in the suppression of somatic HR.Figure 5.


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)

Both N-terminal domains DUF1767 and OB1 have different roles in suppressing HR. The recombination frequencies of (A) untreated and (B) cisplatin (3 µM) treated plants. In contrast to the RMI1ΔDUF and RMI1ΔOB1 constructs, the expression of the recombinant RMI1ΔDUFΔOB1 construct does not lead to any type of complementation of the hyper-recombination phenotype of the Atrmi1-2 mutant. All experiments n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814364&req=5

gkt730-F5: Both N-terminal domains DUF1767 and OB1 have different roles in suppressing HR. The recombination frequencies of (A) untreated and (B) cisplatin (3 µM) treated plants. In contrast to the RMI1ΔDUF and RMI1ΔOB1 constructs, the expression of the recombinant RMI1ΔDUFΔOB1 construct does not lead to any type of complementation of the hyper-recombination phenotype of the Atrmi1-2 mutant. All experiments n = 3.
Mentions: The previous results indicated that both N-terminal domains DUF1767 and OB1 have some function in the suppression of somatic HR. However, both could be involved in the same type of reaction that would require AtRMI1 for some but not all forms of HR suppression detected by our assay system. On the other hand, both domains might contribute, at least partially, in an independent way to the suppression phenotype so that the loss of both domains would lead to a complete loss of suppression. To discriminate between these possibilities, we generated a construct missing both the DUF1767 domain and the OB-fold domain 1 (Figure 2). After transformation of the Atrmi1-2 mutant, four different independent transgenic lines were isolated and analysed. The expression of the recombinant protein RMI1ΔDUFΔOB1 did not significantly change the frequency of HR of the Atrmi1-2 mutant either with or without cisplatin treatment (Figure 5). On the other hand, the HR frequency of all lines was significantly different from that of the wild-type without (#17: P = 0.03; #18: P = 0.02; #19: P = 0.003; #20: P < 0.001, Student’s t-test) or with cisplatin treatment (#17: P = 0.001; #18: P = 0.003; #19: P = 0.004; #20: P = 0.01, Student’s t-test). The fact that Atrmi1 plants expressing an AtRMI1 protein without both the DUF1767 and OB domain 1 show a recombination frequency that is higher than that of plants expressing an AtRMI1 where only one of the domains is missing (compare Figure 5 with Figure 3B and C; Supplementary Figure S3B and C) indicates that both domains have at least partially non-overlapping functions in the suppression of somatic HR.Figure 5.

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