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Published on: September 17, 2020
Isolation and characterization of novel xrs2 mutations in Saccharomyces cerevisiae
Hiroki Shima1, Masakatu Suzuki, Miki Shinohara
1Department of Radiation Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan.
Abstract:
The Mre11/Rad50/Xrs2 (MRX) complex is involved in DNA damage repair, DNA damage response, telomere control, and meiotic recombination. Here, we constructed and characterized novel mutant alleles of XRS2. The alleles with mutations in the C-terminal conserved domain of Xrs2 were grouped into the same class. Mutant Xrs2 in this class lacked Mre11 interaction ability. The second class, lacking a C-terminal end, showed defects only in telomere control. A previous study showed that this C-terminal end contains a Tel1-association domain. These results indicate that Xrs2 contains two functional domains, Mre11- and Tel1-binding domains. While the Mre11-binding domain is essential for Xrs2 function, the Tel1-binding domain may be essential only for Tel1 function in telomere maintenance. The third class, despite containing a large deletion in the N-terminal region, showed no defects in DNA damage repair. However, some mutants, which showed a reduced level of Xrs2 protein, were partially defective in formation of meiotic DSBs and telomere maintenance. These defects were suppressed by overexpression of the mutant Xrs2 protein. This result suggests that the total amount of Xrs2 protein is a critical determinant for the function of the MRX complex especially with regard to telomere maintenance and meiotic DSB formation.
Insights
The Xrs2 protein has two key domains: one for Mre11 interaction, crucial for DNA repair, and another for Tel1 binding, important for telomere control. Protein levels also impact MRX complex function.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- The Mre11/Rad50/Xrs2 (MRX) complex plays vital roles in DNA repair, DNA damage response, telomere maintenance, and meiotic recombination.
- Understanding the functional domains of the XRS2 gene is crucial for elucidating the MRX complex's mechanisms.
Purpose of the Study:
- To construct and characterize novel mutant alleles of XRS2.
- To identify and define the functional domains of the Xrs2 protein and their roles in DNA repair and telomere control.
Main Methods:
- Construction and characterization of XRS2 mutant alleles.
- Analysis of Mre11 interaction and Tel1 association.
- Assessment of DNA damage repair, telomere control, and meiotic double-strand break (DSB) formation.
Main Results:
- Mutations in the C-terminal conserved domain disrupted Mre11 interaction, classifying them as one functional domain.
- Deletion of the C-terminal end, containing a Tel1-association domain, specifically impaired telomere control.
- N-terminal deletions did not affect DNA repair, but reduced Xrs2 protein levels caused defects in meiotic DSB formation and telomere maintenance, which were suppressed by overexpression.
Conclusions:
- Xrs2 possesses two distinct functional domains: an Mre11-binding domain essential for overall Xrs2 function and a Tel1-binding domain primarily involved in telomere maintenance.
- The total amount of Xrs2 protein is a critical factor for MRX complex function, particularly in telomere maintenance and meiotic DSB formation.
