Cdc13Np interacts with Est1Cp in a two-hybrid assay. (A) Two hybrid analysis demonstrates that Cdc13Np interacted with Est1Cp but not with full-length Est2p. Positive and negative controls are the same as in Figure 2A. (B) Ten-fold serial dilutions of each strain were plated onto media selecting for the two-hybrid interaction (Gal - Leu) or selecting for the two plasmids (YC-His-Trp). The terminal 600 amino acids from CDC13, cdc13-1, or cdc13-2 were tested for their ability to interact with Est1Cp.

Polypeptides used in the two-hybrid assay. Dotted regions indicated the portion of Cdc13p expressed from the bait plasmid pEG202 and the portions of proteins expressed from the prey vector pJG4-5 (not to scale). The fusion of the amino-terminal region of Cdc13p with the LexA DNA binding domain is referred to as Cdc13Np in the text. Prey proteins are similarly named; e.g., Pol1Np refers to the fusion of 379 amino acids from near the amino-terminus of Pol1p to the activation domain peptide. The amino acid numbers after the name of each protein indicates the specific amino acids expressed from the two-hybrid vector. The numbers to the right of the schematic for each protein indicate the number of amino acids in the full-length protein.

Analysis of pol1 alleles that disrupt interaction of Pol1p with Cdc13p. (A) Positions of functional domains and locations of various mutations within Pol1p are indicated. Mutations in bold disrupted Pol1p interaction with Cdc13p. Positions or regions containing mutations characterized in other labs are also shown: hpr3 (G439E), cdc17-2 (G637D), cdc17-1 (G904D) (Pizzagalli et al. 1988; Lucchini et al. 1990). (B) The wild-type (lane 1) or mutant (lanes 2–7) proteins were expressed from the pJG4-5 vector under control of a GAL1 promoter. The allele number of the expressed protein is indicated above the lanes. Proteins expressed from pJG4-5 are fused to a transcriptional activation domain and an HA epitope. Cell extracts were analyzed from two different isolates for each mutation using Western blotting with an anti-HA antibody. (C) Extracts were produced from strains expressing both Cdc13Np and either the pJG4-5 vector alone or pJG4-5 with the wild-type POL1 segment or one of the point mutants, as indicated below. Data are the average of three independent β-galactosidase measurements. Error bars, s.d.

Alleles of CDC13 that disrupt interactions with POL1. (A) The portion of Cdc13p that was used as bait in the two-hybrid screen is represented by the dotted region. Asterisks mark the sites of single amino acid substitutions that reduced the interaction of Cdc13Np with Pol1Np by two hybrid criteria. The alleles in bold are those used for phenotypic analyses. The locations of the cdc13-1 and cdc13-2 alleles are also indicated (+) (Lin and Zakian 1996; Nugent et al. 1996). (B) Alleles for cdc13 polypeptides that lost interaction with Pol1Np are named by the number of the mutated residue. The amino acid change in each allele is noted in parentheses. Each mutant allele was checked for its ability to interact with both Pol1Np and Fun12Mp in the two hybrid assay using the LacZ filter assay. Western blotting was used to determine if strains carrying the mutant allele made wild-type levels of Cdc13p. (NT) Not tested (+++) wild-type levels of interaction or protein expression, (−) no interaction or protein expression; (±, +, and ++) intermediate levels of interaction or protein expression. (C) XhoI digested DNA from wild-type strain or cdc13Δ strains carrying a centromere plasmid with mutant cdc13 alleles (cdc13-50 and cdc13-523) that disrupted interaction of Cdc13Np with Pol1Np was analyzed by Southern blotting. Only the lower portion of the gels is shown. (+) Wild type (−) mutant cdc13 alleles.

The interaction of Cdc13p and Est1p can be detected biochemically and has functional significance. (A) Extracts were prepared from cells expressing HA-tagged wild type (lanes 1,4,6) or mutant Cdc13p (lane 2, cdc13-1; lane 3, cdc13-2) or HA-tagged eiF-5Ap (lane 5). Cells also expressed GST-fused Est1p (lanes 1,2,3,5), the GST polypeptide (lane 4) or no GST protein (lane 6). The total extract was analyzed by Western blotting (left panels) with an anti-HA (top) or anti-GST sera (bottom). The extracts were incubated with glutathione sepharose (GS) beads, and the bound proteins eluted (right panels) and analyzed by Western blotting as described for the unfractionated extract. (B) To determine the effect of Est1Cp overexpression on growth, ten-fold serial dilutions of the CDC13 strain carrying pJG4-5 vector (lines 1,2); cdc13-1 carrying pJG4-5 vector (lines 3,4); cdc13-1 expressing Fun12Mp from the pJG4-5 vector (lines 5,6); cdc13-1 expressing Est1Cp from the pJG4-5 vector (lines 7,8) were plated on galactose minus tryptophan medium and grown at 34°C (left) or glucose minus tryptophan medium and grown at 34°C (middle) or glucose minus tryptophan medium and grown at 25°C (right). Expression of Est1Cp and Fun12Mp was under control of the galactose inducible GAL1 promoter.

Cdc13p interacted with Pol1p, the catalytic subunit of DNA polymerase α. (A) Cells expressing prey and/or bait proteins were streaked on galactose plates lacking leucine (Gal–Leu). Galactose induces expression of prey proteins and growth in the absence of leucine requires interaction of bait and prey polypeptides. Alternatively, cells were streaked on glucose plates lacking leucine (Glu-Leu) where prey proteins are not expressed or glucose plates lacking histidine and tryptophan (Glu-His-Trp), which selects for maintenance of both the prey and bait plasmids but not for interaction of their products. The positive control was cells expressing two subunits of RNA polymerase II, Rpb4p and Rpb7p. The negative controls were cells expressing Cdc13Np and carrying the empty prey vector pJG4-5 (Negative Control–1), cells expressing Pol1Np and carrying the empty bait vector pEG202 (Negative Control-2), and cells expressing Cdc13Np and a LexA::cRafp fusion protein (gift from E. Golemis) (Cdc13Np/cRaf). (B) Extracts from cells carrying an MYC₃-tagged Cdc13p were immunoprecipitated by monoclonal anti-MYC antibody immobilized on protein-A and protein-G beads. The extract prior to immunoprecipitation (Total), the supernatant from the immunoprecipitate (Sup.), and the immunoprecipitate (IP) were analyzed by Western blotting using both anti-MYC and antiPol1p monoclonal antibodies. Although Cdc13p was not visible in the total cell extract in this gel, it was detectable when more protein was loaded. Extracts were prepared from cells containing 3XMYC (lanes 1,2) or untagged (lane 3) Cdc13p. Cells had either the wild-type POL1 (lanes 1,3) or the pol1-236 allele (lane 2). The nonspecific band was detected by the anti-MYC serum.

The pol1 alleles that disrupt Pol1p-Cdc13p interaction cause telomere lengthening. (A) Genomic DNA was isolated from wild type and two independent isolates for each of the three pol1 alleles (allele numbers are above the lanes), digested with XhoI and analyzed by Southern hybridization using a telomeric probe. (Lanes 2, 3) pol1-236 cells; (lanes 5, 6) pol1-238 cells; (lanes 8, 9) pol1-241 cells. (Lanes 1, 4, 7, 10) DNA from an otherwise isogenic wild-type strain. (B) Cells of the indicated genotype were streaked on complete media right after meiosis and allowed to form colonies. DNA was prepared from colonies after the first restreak (∼55 divisions postgermination) and after four restreaks (∼130 generations). DNA was analyzed as described for panel A. DNA is from wild type (lanes 1, 4, 7), cdc13-50 cells (lane 2, 55 divisions; lane 3, 155 divisions), and pol1-236 cells (lane 5, 55 divisions; lane 6, 155 divisions). (C) Western analysis was carried out using DNA from POL1 or pol1-236, -238, and -241 strains expressing 3XMYC- Cdc13p (mutant allele indicated above the lanes; extracts from two independent mutant isolates are shown for each mutant allele). Extracts were analyzed by SDS–PAGE and Western blotting using the anti-Pol1p serum (top) or anti-myc serum (lower panel). The minor differences in protein levels were not reproducible. (D) To measure telomere position effect, ten-fold serial dilutions of otherwise isogenic strains that were either wild type (wt) or contained the indicated mutations and having URA3 next to the left telomere of chromosome VII were spotted onto plates containing complete medium plus FOA, complete medium lacking uracil (YC-uracil), or complete medium (YC).