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Functional and genetic analysis of the Saccharomyces cerevisiae RNC1/TRM2: evidences for its involvement in DNA double-strand break repair

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Abstract

We previously isolated the RNC1/TRM2 gene and provided evidence that it encodes a protein with a possible role in DNA double strand break repair. RNC1 was independently re-isolated as the TRM2 gene encoding a methyl transferase involved in tRNA maturation. Here we show that Trm2p purified as a fusion protein displayed 5′ → 3′ exonuclease activity on double-strand (ds) DNA, and endonuclease activity on single-strand (ss) DNA, properties characteristic of previously isolated endo-exonucleases. A variant of Trm2p, Trm2p(ctΔ76aa) lacking 76 amino acids at the C-terminus retained nuclease activities but not the methyl transferase activity. Both the native and the variant exhibited sensitivity to the endo-exonuclease inhibitor pentamidine. The Saccharomyces cerevisiae trm2(Δ232-1920nt) mutant (containing only the first 231 nucleotides of the TRM2 gene) displayed low sensitivity to methyl methane sulfonate (MMS) and suppressed the MMS sensitivity of rad52 mutants in trm2(Δ232-1920nt)rad52 double mutants. The deletion of KU80, in trm2(Δ232-1920nt) mutant background displayed higher MMS sensitivity supporting the view of the possible role of Trm2p in a competing repair pathway separate from NHEJ. In addition, trm2 exo1 double mutants were synergistically more sensitive to MMS and ionizing radiation than either of the single mutant suggesting that TRM2 and EXO1 can functionally complement each other. However, the C-terminal portion, required for its methyl transferase activity was found not important for DNA repair. These results propose an important role for TRM2 in DNA repair with a potential involvement of its nuclease function in homologous recombination based repair of DNA DSBs.

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Acknowledgments

We thank Dr. Tom Wilson and Dr. Alexandre Semionov and Jean-Philip Belzile for helpful discussion and review of this article, Mike Fasulo for providing the plasmid construct pGHOT, and Paul Kauler for his technical assistance. This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Cancer Research Society.

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Authors and Affiliations

  1. Department of Oncology, Faculty of Medicine, McGill University, Montreal General Hospital, 1650 Ave. Cedar, Montreal, Quebec, Canada, H3G 1A4

    Sibgat A. Choudhury, Benyam Asefa, Ashley Webb & Terry Y-K Chow

  2. Centre de recherche, Maisonneuve-Rosemont Hospital, 5415 Boul. L’Assomption, Montreal, Quebec, Canada, H1T 2M4

    Dindial Ramotar

  3. Oncozyme Pharma Inc., Montreal, Quebec, Canada, H1M 1V8

    Terry Y-K Chow

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  1. Sibgat A. Choudhury
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Correspondence to Terry Y-K Chow.

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The authors S. A. Choudhury and B. Asefa contributed equally to the work and manuscript.

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Choudhury, S.A., Asefa, B., Webb, A. et al. Functional and genetic analysis of the Saccharomyces cerevisiae RNC1/TRM2: evidences for its involvement in DNA double-strand break repair. Mol Cell Biochem 300, 215–226 (2007). https://doi.org/10.1007/s11010-006-9386-1

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