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Molecular and Cellular Biology, September 1999, p. 5930-5942, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Active-Site Mutations in the Xrn1p Exoribonuclease of Saccharomyces cerevisiae Reveal a Specific Role in Meiosis

Jachen A. Solinger,1,2 Donatella Pascolini,3 and Wolf-Dietrich Heyer1,2,*

Institute of General Microbiology, University of Bern, CH-3012 Bern,1 and Department of Biochemistry/Sciences II, University of Geneva, CH-1211 Geneva 4,3 Switzerland, and Section of Microbiology, University of California Davis, Davis, California 956162

Received 5 April 1999/Returned for modification 21 May 1999/Accepted 14 June 1999

Xrn1p of Saccharomyces cerevisiae is a major cytoplasmic RNA turnover exonuclease which is evolutionarily conserved from yeasts to mammals. Deletion of the XRN1 gene causes pleiotropic phenotypes, which have been interpreted as indirect consequences of the RNA turnover defect. By sequence comparisons, we have identified three loosely defined, common 5'-3' exonuclease motifs. The significance of motif II has been confirmed by mutant analysis with Xrn1p. The amino acid changes D206A and D208A abolish singly or in combination the exonuclease activity in vivo. These mutations show separation of function. They cause identical phenotypes to that of xrn1Delta in vegetative cells but do not exhibit the severe meiotic arrest and the spore lethality phenotype typical for the deletion. In addition, xrn1-D208A does not cause the severe reduction in meiotic popout recombination in a double mutant with dmc1 as does xrn1Delta . Biochemical analysis of the DNA binding, exonuclease, and homologous pairing activity of purified mutant enzyme demonstrated the specific loss of exonuclease activity. However, the mutant enzyme is competent to promote in vitro assembly of tubulin into microtubules. These results define a separable and specific function of Xrn1p in meiosis which appears unrelated to its RNA turnover function in vegetative cells.

* Corresponding author. Mailing address: Section of Microbiology, University of California, Davis, One Shields Ave., Davis, CA 95616. Phone: (530) 752-3001. Fax: (530) 752-3011. E-mail: wdheyer{at}ucdavis.edu.

Molecular and Cellular Biology, September 1999, p. 5930-5942, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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