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. 2002 Jan 15;30(2):581-91.
doi: 10.1093/nar/30.2.581.

Involvement of rhp23, a Schizosaccharomyces pombe homolog of the human HHR23A and Saccharomyces cerevisiae RAD23 nucleotide excision repair genes, in cell cycle control and protein ubiquitination

Robert T Elder  1 , Xiang-qian Song, Mingzhong Chen, Kevin M Hopkins, Howard B Lieberman, Yuqi Zhao
Affiliations

Involvement of rhp23, a Schizosaccharomyces pombe homolog of the human HHR23A and Saccharomyces cerevisiae RAD23 nucleotide excision repair genes, in cell cycle control and protein ubiquitination

Robert T Elder et al. Nucleic Acids Res. .

Abstract

A functional homolog (rhp23) of human HHR23A and Saccharomyces cerevisiae RAD23 was cloned from the fission yeast Schizosaccharomyces pombe and characterized. Consistent with the role of Rad23 homologs in nucleotide excision repair, rhp23 mutant cells are moderately sensitive to UV light but demonstrate wild-type resistance to gamma-rays and hydroxyurea. Expression of the rhp23, RAD23 or HHR23A cDNA restores UV resistance to the mutant, indicating that rhp23 is a functional homolog of the human and S.cerevisiae genes. The rhp23::ura4 mutation also causes a delay in the G2 phase of the cell cycle which is corrected when rhp23, RAD23 or HHR23A cDNA is expressed. Rhp23 is present throughout the cell but is located predominantly in the nucleus, and the nuclear levels of Rhp23 decrease around the time of S phase in the cell cycle. Rhp23 is ubiquitinated at low levels, but overexpression of the rhp23 cDNA induces a large increase in ubiquitination of other proteins. Consistent with a role in protein ubiquitination, Rhp23 binds ubiquitin, as determined by two-hybrid analysis. Thus, the rhp23 gene plays a role not only in nucleotide excision repair but also in cell cycle regulation and the ubiquitination pathways.

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Figures

Figure 1
Figure 1
The rhp23::ura4 mutant is sensitive to UV light but not ionizing radiation or HU. Cell survival after UV irradiation (A), ionizing radiation exposure (B) or HU treatment (C) of wild-type SP223, rhp23::ura4, rhp23::ura4 (pYZ1N-rph23) and rad9-192 cells. The rad9-192 mutant was used as a control since it is highly sensitive to all three agents (36). The rhp23::ura4 disruption does not cause increased sensitivity to ionizing radiation or HU, but mutant cells are moderately sensitive to UV light in comparison with the resistant wild-type SP223 strain and the highly sensitive rad9-192 mutant strain. The pYZ1N-rhp23 plasmid containing the rhp23 cDNA restores UV resistance to wild-type levels in the rhp23::ura4 disruption strain at both high (no thiamine) and low expression (plus thiamine; data not shown) levels. Experiments in (A) and (B) were performed in medium without thiamine (high expression) and the experiments in (C) were performed in the presence of thiamine (low expression). Error bars represent the standard deviation of three measurements.
Figure 2
Figure 2
Human HHR23A and S.cerevisiae RAD23 restore UV resistance to S.pombe rhp23::ura4. High level expression of RAD23 (A) or HHR23A (B) cDNAs from the pYZ1N plasmid in the S.pombe rhp23::ura4 mutant restores UV resistance. cDNAs in (A) and (B) were expressed under inducing conditions (no thiamine) from the nmt1 promoter (24). In (C) thiamine is present to repress the nmt1 promoter, and at these low expression levels the HHR23A cDNA does not efficiently restore UV resistance to the rhp23::ura4 mutant. Error bars represent the standard deviation of three measurements.
Figure 3
Figure 3
The S.pombe rhp23::ura4 mutation causes a cell cycle G2 delay. Cell length was measured for at least 70 cells during log phase growth in medium with thiamine to repress nmt1 promoter activity. (A) Cell length distribution shown for three strains. (B) The mean cell length shown along with the standard error of the mean.
Figure 4
Figure 4
Rhp23 is located predominantly in the nucleus, and levels of Rhp23 protein are cell cycle dependent. (A) The top three panels show cells containing a single integrated copy of gfp expressed from the nmt1 promoter under inducing conditions. The left panel is a picture of GFP fluorescence; the middle panel illustrates DNA after staining with Hoechst 33342; the right panel shows a differential interference contrast picture of the cells. The bar in the right panel represents 10 µm. The bottom three panels are of cells containing a single integrated copy of the gfp–rhp23 fusion gene under inducing conditions. (B) Wild-type SP223 cells were synchronized in S phase by treatment with HU, an inhibitor of DNA synthesis. After removal of the drug, immunoblots of extracts prepared every 30 min were probed with antibodies against α-tubulin and Rad23. The anti-Rad23 antibodies cross-react with Rhp23 (see Materials and Methods). The lane on the left labeled – is for an extract prepared from the rhp23 deletion strain, and the two lanes on the right labeled + are two dilutions of an extract prepared from a strain overexpressing rhp23 cDNA. The graph represents the amount of Rhp23 relative to α-tubulin determined from scanning the immunoblot. (C) Levels of GFP–Rhp23 in the nucleus decrease at the time of nuclear division. Confocal images of GFP–Rhp23 were taken every 2 min, and the time of the image is shown in each panel. The arrow indicates the cell undergoing nuclear division during this time period.
Figure 5
Figure 5
Rhp23 is ubiquitinated at low levels, and overexpression of rhp23 leads to an accumulation of ubiquitinated proteins. (A) Ubiquitinated forms of Rhp23 are found only in a strain with a mts2 mutation in the proteasome subunit. The molecular weights of size standards (kDa) are indicated to the left of the immunoblot. (B) Overexpression of HA–Rhp23 leads to accumulation of large amounts of ubiquitinated proteins. Cell extracts were prepared from the wild-type strain SP223 without (–) or with (+) a plasmid carrying HA-tagged Rhp23 (HA–Rhp23). Cells were grown in media without thiamine to overexpress the tagged rhp23. Extracts were immunoprecipitated with anti-HA antibodies and the immunoprecipitate (indicated in the IP row by P) and the supernatant (indicated by S) were immunoblotted and probed with anti-ubiquitin (top panel) and anti-Rad23 antibodies (bottom panel). The positions of HA–Rhp23 and the immunoglobin heavy chain (IgHc), which was used as a protein loading control, are indicated. (C) In the left panel, cell extracts from the rhp23::ura4 strain carrying a plasmid with the rhp23, RAD23 or HHR23 cDNA or a vector control were prepared under inducing (+) or repressing (–) conditions for the nmt1 promoter expressing the cDNAs. For all extracts, 120 µg protein per lane were loaded on the gel, except for the induced rhp23 cDNA condition, where dilutions of 120, 12 and 2.4 µg were loaded (lanes 1–3). The upper immunoblot was probed with anti-ubiquitin antibodies. The lower immunoblot was probed with anti-Rad23 antibodies, which cross-react with Rhp23 but do not cross-react with HHR23A. The positions of Rhp23 and the slightly larger Rad23 are indicated. In the right panels, extracts were prepared from the rhp23::ura4 strain carrying a plasmid with the rhp23 cDNA either without (lane 1) or with induction (lanes 2–6). For the induced samples, cycloheximide (Chx) was added to 100 µg/ml at time 0 and samples taken 30, 60, 90 and 120 min later. Immunoblots with 120 µg protein were probed with anti-ubiquitin antibodies (upper panel) and with anti-Rad23 antibodies (lower panel). The samples in the right panel were run on a higher percentage polyacrylamide gel than in the left panel, so the anti-ubiquitin bands appear more compressed in the right panel.
Figure 6
Figure 6
Rhp23-binding proteins. (A) Interacting proteins identified in the yeast two-hybrid system. The β-galacatosidase activity of strains carrying the indicated cDNAs in the yeast two-hybrid system was determined in triplicate by a plate assay and a liquid assay (42). The units column shows the mean and standard deviation for three measurements in the liquid assay. The positive control on the top line is HIV-1 Vpr and human UNG, which interact in the two-hybrid system (49). The negative control on the bottom line is rhp23 with the pGAD GH vector (ClonTech, CA) not containing an insert. (B) Alignment of Rhp23-binding proteins. The amino acid sequences were aligned using a multiple sequence alignment Jellyfish program (Biowire). Identical amino acids are in dark color. The highly similar and moderately similar residues are indicated by light color. Consensus amino acids are listed under each residue. Note that the first 76 amino acids, which correspond to the ubiquitin sequence, are the only homologous sequences shared by all four proteins.
Figure 6
Figure 6
Rhp23-binding proteins. (A) Interacting proteins identified in the yeast two-hybrid system. The β-galacatosidase activity of strains carrying the indicated cDNAs in the yeast two-hybrid system was determined in triplicate by a plate assay and a liquid assay (42). The units column shows the mean and standard deviation for three measurements in the liquid assay. The positive control on the top line is HIV-1 Vpr and human UNG, which interact in the two-hybrid system (49). The negative control on the bottom line is rhp23 with the pGAD GH vector (ClonTech, CA) not containing an insert. (B) Alignment of Rhp23-binding proteins. The amino acid sequences were aligned using a multiple sequence alignment Jellyfish program (Biowire). Identical amino acids are in dark color. The highly similar and moderately similar residues are indicated by light color. Consensus amino acids are listed under each residue. Note that the first 76 amino acids, which correspond to the ubiquitin sequence, are the only homologous sequences shared by all four proteins.
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