Ubc4 and Not4 regulate steady-state levels of DNA polymerase-α to promote efficient and accurate DNA replication

Abstract

The accurate duplication of chromosomal DNA is required to maintain genomic integrity. However, from an evolutionary point of view, a low mutation rate during DNA replication is desirable. One way to strike the right balance between accuracy and limited mutagenesis is to use a DNA polymerase that lacks proofreading activity but contributes to DNA replication in a very restricted manner. DNA polymerase-α fits this purpose exactly, but little is known about its regulation at the replication fork. Minichromosome maintenance protein (Mcm) 10 regulates the stability of the catalytic subunit of pol-α in budding yeast and human cells. Cdc17, the catalytic subunit of pol-α in yeast, is rapidly degraded after depletion of Mcm10. Here we show that Ubc4 and Not4 are required for Cdc17 destabilization. Disruption of Cdc17 turnover resulted in sensitivity to hydroxyurea, suggesting that this pathway is important for DNA replication. Furthermore, overexpression of Cdc17 in ubc4 and not4 mutants caused slow growth and synthetic dosage lethality, respectively. Our data suggest that Cdc17 levels are very tightly regulated through the opposing forces of Ubc4 and Not4 (destabilization) and Mcm10 (stabilization). We conclude that regular turnover of Cdc17 via Ubc4 and Not4 is required for proper cell proliferation.

Figure 1.

Cdc17 degradation is proteasome-dependent. ABy527 (mcm10-1, pdr5Δ, rpn4Δ) cells were grown in rich liquid medium at 25°C to midlog phase and split in four. MG132 was added to two of the cultures, DMSO was added to the other two, and cultures were shifted either to 30°C or 37°C for 180 min. Cdc17–3HA and α-tubulin were analyzed by Western blot.

Figure 2.

Overexpressed Cdc17 is stable in ubc4Δ cells. Strains were grown overnight in minimal medium with 2% raffinose. Cdc17-2HA was overexpressed from a galactose promoter in the presence of 2% galactose for 2 h. Cells were pelleted, resuspended in YPD, and samples were taken at 0, 60, 120, and 180 min after addition of glucose. Cdc17-2HA and α-tubulin, which served as a loading control, were analyzed by Western blot. The graphs show the quantification of Cdc17 relative to α-tubulin at each time point in each mutant strain relative to wild-type. The wild-type data in each graph are from the same experiment.

Figure 3.

Ubc4 is the E2 enzyme required for endogenous Cdc17 degradation in the absence of Mcm10. (A) Asynchronous cultures of ABy448 (W303-1a), ABy013 (mcm10-1), ABy342 (mcm10-1, ubc4Δ), ABy363 (mcm10-1, ubc5Δ), and ABy359 (mcm10-1, rad6Δ) grown at 25°C were shifted to 37°C for 180 min. Cdc17–3HA and α-tubulin were analyzed by Western blot. (B) The graph shows Cdc17/tubulin ratios at each time point for each strain relative to wild-type at time 0 (average of 3 separate experiments, bars represent mean ± SD).

Figure 4.

Not4 is the E3 ligase required for endogenous Cdc17 degradation in the absence of Mcm10. (A) Asynchronous cultures of ABy013 (mcm10-1), ABy342 (mcm10-1, ubc4Δ), and ABy529 (mcm10-1, not4Δ) grown at 25°C were shifted to 37°C for 180 min. Cdc17–3HA and α-tubulin were analyzed by Western blot. (B) The graph shows Cdc17/tubulin ratios at each time point for each strain relative to mcm10-1 at time 0 (average of 3 separate experiments, bars represent mean ± SD). (C) Asynchronous cultures of ABy781 (mcm10-1, pRS316), ABy762 (mcm10-1, not4Δ, pRS316), ABy738 (mcm10-1, not4Δ, pRS316-NOT4), and ABy739 (mcm10-1, not4Δ, pRS316-not4-L35A) grown at 25°C were shifted to 37°C for 180 or 360 min. Cdc17–3HA and α-tubulin were analyzed by Western blot. (D) The graph shows Cdc17/tubulin ratios at each time point for each strain relative to ABy781 at time 0 (average of 3 separate experiments, bars represent mean ± SD). (E) Asynchronous cultures of ABy596 (mcm10-1, NOT4-3HA), ABy740 (mcm10-1, not4Δ, pRS316-NOT4-3HA), and ABy741 (mcm10-1, not4Δ, pRS316-not4-L35A-3HA) were grown in SC-ura liquid medium at 25°C to midlog phase. Not4-3HA and α-tubulin were analyzed by Western blot. (F) ABy661 (mcm10-1, pdr5Δ, rpn4Δ, YEp105) and ABy1051 (mcm10-1, pdr5Δ, rpn4Δ, YEp105-G75,76A) were grown in rich medium to OD₆₀₀ = 0.6 at 25°C and shifted to 37°C for 3 h. Total protein was precipitated with trichloroacetic acid and analyzed by Western blot using anti-HA and anti-tubulin antibodies.

Figure 5.

Cdc17 is degraded primarily in the nucleus. (A) ABy150 (S288C), ABy152 (ubc4Δ), ABy574 (UBC4-NES-3HA), ABy577 (ubc5Δ, UBC4-NES-3HA), and GAP510 (ubc4Δ, ubc5Δ) were heat shocked as described in the Materials and Methods, aliquots were grown on YPD plates for 3 d at 30°C, and colonies were counted to determine the percentage of cells that were viable following heat shock. (B) ABy605 (mcm10-1, UBC4-GFP) and ABy660 (mcm10-1, UBC4-NES-GFP) were examined using fluorescence microscopy. Merge represents GFP and DAPI. Yellow dots represent colocalization of Ubc4-GFP and DAPI-stained nuclei. Red dots represent DAPI-stained nuclei in which Ubc4-GFP is not present. Black circles represent vacuoles. (C) Asynchronous cultures of ABy013 (mcm10-1), ABy342 (mcm10-1, ubc4Δ), ABy455 (mcm10-1, UBC4-NES-3HA), ABy363 (mcm10-1, ubc5Δ), and ABy359 (mcm10-1, rad6Δ) grown at 25°C were shifted to 37°C for 180 min. Cdc17–3HA and α-tubulin were analyzed by Western blot. (D) The graph shows Cdc17/tubulin ratios at each time point for each strain relative to mcm10-1 at time 0 (average of 3 separate experiments, bars represent mean ± SD).

Figure 6.

CDC17 mRNA levels are unchanged in not4Δ mutants. ABy448 (W303-1a), ABy013 (mcm10-1), ABy342 (mcm10-1, ubc4Δ), ABy455 (mcm10-1, UBC4-NES), ABy529 (mcm10-1, not4Δ), ABy363 (mcm10-1, ubc5Δ), ABy359 (mcm10-1, rad6Δ), ABy360 (mcm10-1, ubc13Δ), and ABy361 (mcm10-1, mms2Δ) were grown in rich medium to midlog phase at 25°C and shifted to 37°C for 180 min. Total RNA was extracted from cells taken just before the shift to 37°C and after 180 min at 37°C. Semiquantitative RT-PCR was performed on each sample using 30, 6, and 3 ng total RNA with primers specific for either CDC17 or ACT1.

Figure 7.

Cdc17 levels are elevated twofold in the presence of Mcm10 in ubc4Δ, UBC4-NES-3HA, and not4Δ cells. (A) Asynchronous cultures of ABy013 (mcm10-1), ABy342 (mcm10-1, ubc4Δ), ABy455 (mcm10-1, UBC4-NES-3HA), ABy363 (mcm10-1, ubc5Δ), ABy359 (mcm10-1, rad6Δ), ABy360 (mcm10-1, ubc13Δ), ABy361 (mcm10-1, mms2Δ), and ABy529 (mcm10-1, not4Δ) were grown at 25°C to midlog phase. Cdc17–3HA and α-tubulin were analyzed by Western blot. (B) The graph shows the fold change in Cdc17 in each strain relative to mcm10-1 (average of 3 separate experiments, bars represent mean ± SD). (C) Asynchronous cultures of ABy448 (W303-1a), ABy607 (ubc4Δ), ABy597 (not4Δ), and ABy764 (ubc5Δ) were grown at 25°C to midlog phase. Cdc17–3HA and α-tubulin were analyzed by Western blot. The values represent the fold change in Cdc17 levels relative to wild-type.

Figure 8.

Ubc4 is required in the nucleus for tolerance to hydroxyurea. (A) Successive 10-fold dilutions of ABy448 (W303-1a), ABy607 (ubc4Δ), ABy662 (UBC4-NES-3HA), ABy597 (not4Δ), ABy764 (ubc5Δ), ABy013 (mcm10-1), ABy342 (mcm10-1, ubc4Δ), ABy455 (mcm10-1, UBC4-NES-3HA), ABy529 (mcm10-1, not4Δ), and ABy363 (mcm10-1, ubc5Δ) were grown for 3 d at 30°C on YPD plates and YPD containing 50 mM, 100 mM, and 200 mM HU. (B) Asynchronous cultures of ABy597 (not4Δ) and ABy529 (mcm10-1, not4Δ) were grown to midlog phase. Cdc17–3HA and α-tubulin were analyzed by Western blot in undiluted extracts and extracts diluted 10-fold, 25-fold, and 50-fold.

Figure 9.

Overexpression of Cdc17 and Mcm10 causes slow growth. (A) Strains containing the indicated plasmids were grown in media containing 2% raffinose to midlog phase, either 2% galactose (GAL) or 2% glucose (GLU) was added, and cells were grown for an additional 2 h. Cdc17-2HA, Mcm10-3HA, Pol12–3HA, Pri1–3HA, and Pri2–3HA were analyzed by Western blot. (B) Successive 10-fold dilutions of ABy695 (empty vectors), ABy694 (GAL-CDC17-2HA), ABy696 (GAL-MCM10-3HA), ABy705 (GAL-CDC17-2HA, GAL-MCM10-3HA), and ABy732 (GAL-CDC17-2HA, GAL-MCM10-3HA, GAL-POL12–3HA, GAL-PRI1–3HA, GAL-PRI2–3HA) were grown for 3 d at 30°C on SC-his-trp-ura with 2% glucose plates and SC-his-trp-ura with 2% galactose plates.

Figure 10.

Overexpression of Cdc17 and Mcm10 increases genome instability. (A) Seven individual colonies of ABy563 (pRS426gal-CDC17-2HA), ABy564 (pRS423gal-MCM10-3HA), and ABy565 (pRS423gal-MCM10-3HA, pRS426gal-CDC17-2HA) were grown to saturation in the presence of 2% galactose, and seven colonies of ABy565 were grown to saturation in the presence of 2% glucose. Cells were grown on -arg plates containing canavanine (60 mg/L) to select for mutants and on YPD plates for a viable cell count. Colonies were counted after 2–3 d at 30°C. (B) Strains containing the indicated plasmids were grown in media containing 2% raffinose to midlog phase, either 2% galactose (GAL) or 2% glucose (GLU) was added, and cells were grown for an additional 2 h. Cdc17-2HA and Mcm10-3HA were analyzed by Western blot. (C) Seven individual colonies each from ABy640, ABy641, ABy642, and ABy643 were grown in SC-his-trp-leu with 2% galactose to saturation at 30°C. From each culture, 100 cells were plated on SC-trp with 2% glucose medium and 10⁷ cells were plated on SC-trp with 2% glucose and 1 mg/ml 5-FOA medium. Plates were incubated for 5 d at 30°C and colonies were counted. (D) pSH44 was isolated from 5-FOA-resistant clones and digested with HindIII, which produces a 4.3-kb fragment (backbone) and a 3.6-kb fragment [poly(GT)-URA3 coding sequence] in the wild type plasmid. Digested plasmids showed either no detectable size change (circles) or complex rearrangements (squares). (E) Same as in C, but with the addition of strains ABy977, ABy978, ABy979 and ABy980, which contain pRS316-TRP1 in place of pSH44.

Figure 11.

Overexpression of Cdc17 in ubc4Δubc5Δ and not4Δ mutants causes synthetic dosage lethality. (A) ABy962 (pRS423gal), ABy963 (GAL-CDC17-2HA), ABy964 (GAL-MCM10-3HA), ABy965 (ubc4Δ, pRS423gal), ABy966 (ubc4Δ, GAL-CDC17-2HA), ABy967 (ubc4Δ, GAL-MCM10-3HA), ABy968 (ubc5Δ, pRS423gal), ABy969 (ubc5Δ, GAL-CDC17-2HA), ABy970 (ubc5Δ, GAL-MCM10-3HA), ABy971 (ubc4Δubc5Δ, pRS423gal), ABy972 (ubc4Δubc5Δ, GAL-CDC17-2HA), ABy973 (ubc4Δubc5Δ, GAL-MCM10-3HA), ABy974 (not4Δ, pRS423gal), ABy975 (not4Δ, GAL-CDC17-2HA), and ABy976 (not4Δ, GAL-MCM10-3HA) were grown in media containing 2% raffinose to midlog phase, either 2% galactose (GAL) or 2% glucose (GLU) was added, and cells were grown for an additional hr. Cdc17-2HA, Mcm10-3HA, and α-tubulin were analyzed by Western blot. The asterisk indicates a nonspecific band recognized by the HA antibody. (B) Successive 10-fold dilutions of the strains in A were grown for the indicated time at 30°C on SC-his-trp-ura with 2% glucose plates and SC-his-trp-ura with 2% galactose plates. (C) ABy981 (CDC17-3HA, pRS426gal-CDC17-2HA), ABy831 (ubc4Δ, CDC17–3HA, pRS426gal-CDC17-2HA), and ABy837 (not4Δ, CDC17–3HA, pRS426gal-CDC17-2HA) were grown overnight in SC-ura medium to midlog phase. Cdc17 and α-tubulin were analyzed by Western blot.

Figure 12.

Overexpression of Mcm10 and Cdc17 may hinder polymerase switching. After pol-α extends the RNA primer (red line) by 12–20 nucleotides, Mcm10 is ubiquitinated at two distinct lysines (yellow star; (Das-Bradoo et al., 2006). PCNA is recruited to the lagging strand, and a polymerase switch occurs between pol-α and pol-δ, after which pol-α is released from ubiquitinated Mcm10 and recognized by Ubc4 and Not4. The question mark denotes the fact that it is currently unclear whether the interaction is direct or indirect. Accumulation of Cdc17 and Mcm10 interferes with the switch to pol-δ, allowing pol-α to synthesize more DNA than in wild-type cells.