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Eukaryotic Cell, April 2005, p. 832-835, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.832-835.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

Terri S. Rice,1 Min Ding,2 David S. Pederson,2 and Nicholas H. Heintz1*

Departments of Pathology,1 Microbiology and Molecular Genetics, Vermont Cancer Center, University of Vermont College of Medicine, Burlington, Vermont 054052

Received 28 September 2004/ Accepted 21 January 2005


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ABSTRACT
 
Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.


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TEXT
 
In eukaryotes, DNA replication is linked to other cell cycle processes through the stepwise assembly of replication initiation complexes (1, 5, 13). Initiator function is provided by the origin recognition complex (ORC), which binds to replication origins and recruits factors required for assembly of prereplication complexes, a process known as origin licensing (3). ORC and many aspects of origin licensing are conserved in eukaryotes (7, 11). ORC also functions in heterochromatin-induced silencing of transcription in yeast (2, 6) and Drosophila melanogaster (8) and in chromosome condensation and segregation (16, 17).

ORC interacts with other factors, including the DNA binding proteins AIF-C1 and AIF-C2 (20), the transcription factor c-Myc (22), cyclin B/cdc2 complexes (24), and the HBO1 histone acetyltransferase (10). These and other interactions provide avenues for ORC to influence chromatin structure (10). Here we show ORC interacts with Thg1p, the guanylyltransferase of Saccharomyces cerevisiae required for the addition of the G1 residue to the 5' end of histidine tRNA (9).

To identify proteins that interact with ORC, hemagglutinin (HA)-tagged Orc2p was fused to the DNA binding domain of Gal4p and used as bait in a two-hybrid screen as described previously (26). Y190 cells harboring the bait plasmid pAS1-ORC2 were transformed with an S. cerevisiae cDNA expression library; out of 2.6 x 106 transformants, 55 survived selection for histidine prototrophy and ß-galactosidase activity. After recovery in Escherichia coli, candidate plasmids were tested for specificity by mating to Y190 cells that contained either pAS1-ORC2 or unrelated bait plasmids. Eight of 55 plasmids encoded Thg1p (9), and ß-galactosidase assays indicated that Thg1p interacts with Orc2p but not p53, Cdk2, PCNA, Cdc6, Snf1, or lamin (Fig. 1A). Gene disruption experiments showed that THG1 is essential for viability in S. cerevisiae (Fig. 1B), consistent with other reports (23). Incubation of 35S-methionine-labeled Orc2p with beads loaded with glutathione S-transferase (GST), GST-Y4 (Y4 control protein) or GST-scThg1p revealed weak but specific binding of Orc2p to GST-scThg1p (Fig. 1C, lane 7). This specific but weak interaction was also observed in coimmunoprecipitation assays (Fig. 1D, lane 4). Although tests for synthetic lethality between thg1(Ts) and orc2(Ts) yeast strains were negative (data not shown), overexpression of HA-Thg1p markedly suppressed the growth of orc2(Ts) cells at the permissive temperature but not that of wild-type W303, orc5(Ts), cdc7(Ts), or cdc28(Ts) strains (Fig. 2A). The pattern of HA-Thg1p expression in orc2(Ts) cells also differed significantly from that in wild-type, orc5(Ts), or cdc7(Ts) strains (Fig. 2B). These differences were not dependent upon cell cycle arrest or cell death since they were not observed in orc5(Ts) or cdc7(Ts) strains at the restrictive temperature and were present in orc2(Ts) cells at both permissive and restrictive temperatures.



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  		FIG. 1. S. cerevisiae Thg1p interacts with ORC and is essential for viability. (A) S. cerevisiae open reading frame YRG024c was isolated in a yeast two-hybrid screen using Orc2p as bait. YGR024c encodes Thg1p, a histidine tRNA guanylyltransferase. The two-hybrid interaction between Thg1p and Orc2p is indicated by expression of ß-galactosidase, producing a blue color on X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) plates. (B) Tetrad dissection of MD102 (THG/thg1::URA3) showed a two live-two dead segregation pattern, indicating that THG1 is essential for viability in S. cerevisiae. (C) GST-tagged S. cerevisiae Thg1p interacts with Orc2p in a GST pull-down assay. GST alone, GST-Y4, and GST-Thg1p bound to glutathione Sepharose beads, and beads alone were incubated with 35S-labeled Orc2p. After washing, the bound protein fraction (BD) was compared to the flowthrough fraction (FL) by gel electrophoresis and autoradiography. (D) Coimmunoprecipitation of HA-Thg1p with Orc2p. Whole-cell extracts (WCE; 0.5 mg of protein) from yeast strain MD105 [thg1::URA3p(UASgalHA-THG1)] were immunoprecipitated with antibodies to Orc2 or E2F1 as indicated, and the immunoprecipitates (IP) then were probed for HA-Thg1p with 12CA5 antibody. HA-Thg1p (32 kDa) was recovered more efficiently in samples immunoprecipitated with antibodies to Orc2p (lane 4) than E2F1 (lane 5) or extracts incubated with protein A beads alone (lane 6). Background bands of immunoglobulin G heavy chain were observed at 50 to 55 kDa in all samples containing antibodies.



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  		FIG. 2. Overexpression of HA-Thg1p in orc2-1(Ts) cells inhibits cell growth. (A) HA-Thg1p was expressed under the control of the galactose promoter in strains bearing temperature-sensitive alleles for the indicated replication factor; expression of Clf1p was used as a control. At 23°C each test strain grew well on plates containing glucose, whereas on plates containing galactose and raffinose (Gal plus Raf) expression of Thg1p-HA caused a reduction in growth of the orc2-1(Ts) strain, but not W3031a (WT), orc5-1(Ts), cdc7-1(Ts), or cdc28-1(Ts) strains. At 37°C none of the temperature-sensitive strains grew on plates containing either glucose or galactose plus raffinose. The pie chart on the right provides a key to yeast strains. (B) Cell extracts were prepared from the indicated strains containing or lacking pGAL-HA-Thg1 and probed for expression of HA-Thg1p by immunoblotting with anti-HA monoclonal antibody 12CA5. The protein expression pattern of HA-Thg1p in orc2-1(Ts) cells differed from that observed in WT, orc5-1(Ts) or cdc7-1(Ts) yeast strains at both permissive and nonpermissive temperatures.

We next subjected THG1 to random mutagenesis (15) and isolated several temperature-sensitive alleles. When shifted to the nonpermissive temperature (37°C), cells containing the thg1-28(Ts) allele under the control of the endogenous THG1 promoter (19) showed a normal pattern of actin polarization to daughter buds during initial phases of bud formation at the restrictive temperature (data not shown). With time, however, SYBR Green staining for DNA content (14) revealed that most cells collected in the G2/M phase of the cell cycle (Fig. 3B). After 3 h at the nonpermissive temperature many mutant cells displayed short thick bundles of tubulin (data not shown), suggesting that these cells were arrested in either prophase or G2 (12), where the intranuclear spindle is shorter than the metaphase spindle (21). thg1(Ts)HA cells held at the semipermissive temperature of 33°C developed multibudded cells with markedly abnormal bud morphology (Fig. 3C). Staining for chitin with calcofluor (18) showed mother cells with as many as three to four daughter buds (Fig. 3D), indicating that new buds had emerged from daughter cells before cytokinesis had separated mother-daughter progenitor pairs.



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  		FIG. 3. Terminal phenotypes of thg1-28(Ts) S. cerevisiae cells. (A, B) Thg1p is required for the G2/M transition. thg1(Ts)HA cells were propagated at the permissive (A) or nonpermissive (B) temperature. At the indicated times aliquots of cells from asynchronous cultures were stained for DNA content with SYBR Green and analyzed by flow cytometry to determine their distribution in the cell cycle. The majority of thg(Ts)HA cells held at the nonpermissive temperature of 37°C accumulated in G2/M by 7.5 h (2.5 generations). (C and D) Thg1(Ts)HA cells were propagated at the semipermissive temperature of 33°C for 16 h and then examined by phase microscopy (C) or stained with calcofluor to visualize chitin rings (D). (E-H) Thg1p is required for nuclear division and migration. Comparison of phase micrographs (E, G) with cells stained with SYBR Green (F) or DAPI (H) for DNA showed that Thg1(Ts)HA cells exhibit a defect in the segregation of DNA, resulting in mother cells with one or more buds but no DNA (arrows).

Staining of thg1(Ts)HA mutants with SYBR Green or 4,6-diamidino-2-phenylindole (DAPI) also revealed an unusual distribution of DNA in cells held at the nonpermissive temperature; in ~20% of multibudded cells mother cells completely lacked nuclear DNA, whereas daughter buds were positive for nuclear DNA (Fig. 3E to H). In many multibudded cells, the nucleus failed to divide but nonetheless was pulled into the daughter bud by an intact intranuclear spindle. This appears to have been followed by a new round of bud formation. Thus, the budding cycle that initiates at Start continues for multiple cell cycles at the nonpermissive temperature in thg1(Ts)HA cells but is uncoupled from nuclear division and migration. Remarkably, thg1(Ts)HA cells held at the restrictive temperature were viable, for multibudded thg1(Ts)HA cells incubated for 16 h at 37°C in liquid culture formed colonies with the same efficiency as the control strain when plated and incubated at the permissive temperature (data not shown). thg1(Ts)HA cells did not show increased sensitivity to UV light, the ribonucleotide reductase inhibitor hydroxyurea, or the microtubule depolymerizing drug benomyl at 25, 30, or 33.5°C (data not shown).

Previously we showed that Clf1p, a factor required for pre-mRNA splicing, associates with replication origins in an ORC-dependent manner (26). Noc3p, a protein required for pre-rRNA processing, and Yph1p, a protein required for biogenesis of 60S ribosomal subunits, have also been reported to interact with ORC (4, 25). Here we link a factor involved in regulation of histidine tRNA biosynthesis to ORC, establishing an important connection between basic aspects of cell metabolism and cell cycle progression, perhaps in this instance through the nucleotide GTP. The Thg1p coding sequence is very highly conserved (9), and expression of the human protein rescues growth of yeast cells lacking THG1 as well as the yeast protein does (data not shown). We are presently investigating whether expression of TGH1 is required for progression through G2/M in human cells.


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ACKNOWLEDGMENTS
 
T.S.R. was supported by a training grant from the National Institute of Environmental Health Sciences. M.D. was supported in part by a grant from the National Institutes of Health (GM-52017) to D.S.P.

We thank Steven Elledge for two-hybrid reagents, Hiroko Hata for the hsTHG1 plasmid, and Bruce Stillman for Orc2 antibodies.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405. Phone: (802) 656-0372. Fax: (802) 656-8892. E-mail: Nicholas.Heintz{at}uvm.edu. Back


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Eukaryotic Cell, April 2005, p. 832-835, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.832-835.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jackman, J. E., Phizicky, E. M. (2006). tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition. Proc. Natl. Acad. Sci. USA 103: 8640-8645 [Abstract] [Full Text]  
  • Gu, W., Hurto, R. L., Hopper, A. K., Grayhack, E. J., Phizicky, E. M. (2005). Depletion of Saccharomyces cerevisiae tRNAHis Guanylyltransferase Thg1p Leads to Uncharged tRNAHis with Additional m5C. Mol. Cell. Biol. 25: 8191-8201 [Abstract] [Full Text]  

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