Evidence That the pre-mRNA Splicing Factor Clf1p Plays a Role in DNA Replication in Saccharomyces cerevisiae

Evidence That the pre-mRNA Splicing Factor Clf1p Plays a Role in DNA Replication in Saccharomyces cerevisiae

Wenge Zhu^a,c, Irene R. Rainville^a,c, Min Ding^a, Margaret Bolus^a, Nicholas H. Heintz^a,b,c, and David S. Pederson^a,c,d
^a Departments of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405-0068
^b Pathology, University of Vermont, Burlington, Vermont 05405-0068
^c Cell and Molecular Biology Program, University of Vermont, Burlington, Vermont 05405-0068
^d Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405-0068

Corresponding author: David S. Pederson, Stafford Bldg., Rm. 302, 95 Carrigan Dr., University of Vermont, Burlington, VT 05405-0084., dpederso{at}zoo.uvm.edu (E-mail)

Communicating editor: J. RINE

Clf1p is an essential, highly conserved protein in S. cerevisiaethat has been implicated in pre-mRNA splicing. Clf1p's orthologin Drosophila, Crn, is required for normal cell proliferation.Cells depleted of Clf1p arrest primarily with large buds, asingle nucleus, a 2C DNA content, and a short, intact mitoticspindle. We isolated temperature-sensitive clf1 mutants thatexhibit similar mitotic defects when released to the restrictivetemperature from an early S-phase block. While these mutantsalso accumulate unspliced pre-mRNA at the restrictive temperature,the mitotic arrest does not appear to result from a failureto splice tubulin pre-mRNA. Moreover, the same mutants exhibita delayed entry into S phase when released to the restrictivetemperature from a G1 phase block. This delay could not be suppressedby disruption of the S-phase CDK inhibitor SIC1, suggestingthat Clf1p is involved in DNA replication. Consistent with thispossibility, we find that Clf1p (but not the mutant clf1p) interactswith the DNA replication initiation protein Orc2p in two-hybridand co-immunoprecipitation assays, that Clf1p preferentiallyassociates with origins of DNA replication, and that this associationis Orc2p dependent. These observations suggest that Clf1p playsa direct role in the initiation of DNA replication.

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