DEG1, encoding the tRNA:pseudouridine synthase Pus3p, impacts HOT1-stimulated recombination in Saccharomyces cerevisiae
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In Saccharomyces cerevisiae, HOT1-stimulated recombination has been implicated in maintaining homology between repeated ribosomal RNA genes. The ability of HOT1 to stimulate genetic exchange requires RNA polymerase I transcription across the recombining sequences. The trans-acting nuclear mutation hrm3-1 specifically reduces HOT1-dependent recombination and prevents cell growth at 37°. The HRM3 gene is identical to DEG1. Excisive, but not gene replacement, recombination is reduced in HOT1-adjacent sequences in deg1Δ mutants. Excisive recombination within the genomic rDNA repeats is also decreased. The hypo-recombination and temperature-sensitive phenotypes of deg1Δ mutants are recessive. Deletion of DEG1 did not affect the rate of transcription from HOT1 or rDNA suggesting that while transcription is necessary it is not sufficient for HOT1 activity. Pseudouridine synthase 3 (Pus3p), the DEG1 gene product, modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine. Cells deficient in pseudouridine synthases encoded by PUS1, PUS2 or PUS4 displayed no recombination defects, indicating that Pus3p plays a specific role in HOT1 activity. Pus3p is unique in its ability to modulate frameshifting and readthrough events during translation, and this aspect of its activity may be responsible for HOT1 recombination phenotypes observed in deg1 mutants.
KeywordsHOT1 Recombination hotspot DEG1 tRNA:pseudouridine synthase Pus3p
We thank Susan DiBartolomeis, Tom Reiner, Laura Palmer and Reeta Prusty for sharing their valuable technical expertise, and Anita Hopper for critical reading of the manuscript. Technical assistance from Jonathan Morgan and Paul Lewis is also appreciated. Plasmids containing deg1-D151A and controls were kindly provided by Henri Grosjean (CNRS, Laboratoire d‘Enzymologie et de Biochimie Structurales, France). This research was supported by funding from the Pennsylvania State System of Higher Education Faculty Professional Development Council and the Millersville University Faculty Grants Committee to CEH, a Sigma Xi Grant-in-Aid of Research to KGS, Millersville University Alumni Association Neimeyer-Hodgson Student Research Grants to MM, AR, GIL and DLH, and by National Institutes of Health grant GM-36422 to RLK.
- Björk GR (1995) Biosynthesis and function of modified nucleosides in tRNA. In: Söll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. ASM Press, Washington, pp 165–205Google Scholar
- Roeder GS, Keil RL, Voelkel-Meiman KA (1986) A recombination-stimulating sequence in the ribosomal RNA gene cluster of yeast. In: Klar A, Strathern JN (eds) Mechanisms of yeast recombination. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 29–33Google Scholar
- Rose MD, Winston F, Hieter P (1990) Methods in Yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Wai H, Johzuka K, Vu L, Eliason K, Kobayashi T, Horiuchi T, Nomura M (2001) Yeast RNA polymerase I enhancer is dispensable for transcription of the chromosomal rRNA gene and cell growth, and its apparent transcription enhancement from ectopic promoters requires Fob1 protein. Mol Cell Biol 21:5541–5553PubMedCrossRefGoogle Scholar