Nuclear distribution and chromatin association of DNA polymerase alpha-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast

Abstract

Background: Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.

Results: Insertion of a TEV protease cleavage site into Cdc23 allows in vivo removal of the C-terminal 170 aa of the protein by TEV protease induction, resulting in an S phase arrest. This C-terminal fragment of Cdc23 is not retained in the nucleus after cleavage, showing that it lacks a nuclear localization signal and ability to bind to chromatin. Using an in situ chromatin binding procedure we have determined how the S phase chromatin association of DNA polymerase alpha-primase and the GINS (Sld5-Psf1-Psf2-Psf3) complex is affected by Cdc23 inactivation. The chromatin binding and sub-nuclear distribution of DNA primase catalytic subunit (Spp1) is affected by Cdc23 cleavage and also by inactivation of Cdc23 using a degron allele, implying that DNA polymerase alpha-primase function is dependent on Cdc23. In contrast to the effect on Spp1, the chromatin association of the Psf2 subunit of the GINS complex is not affected by Cdc23 inactivation.

Conclusion: An important function of Cdc23 in the elongation step of DNA replication may be to assist in the docking of DNA polymerase alpha-primase to chromatin.

Figure 1

Effects of TEV protease expression on wild-type and mcm7-GFP_N760::Tcs S. pombe strains. A. Western analysis comparing levels of expression of TEV protease in strain P1292 in – thiamine medium (lane 2) and + thiamine medium (lane 3). Lane 1 is protein extract from a strain lacking the pREP3X-TEV-NLS plasmid (P138). B. Expression of TEV protease does not affect the viability of S. pombe. Serial dilutions of strain P1292 were spotted onto plus (+T) and minus (-T) medium and incubated at the temperatures shown. C. Design of Mcm7-GFP_N760::Tcs. The Mcm7 gene was modified so that a TEV cleavage site, fused to GFP is expressed after the last codon of the Mcm7 reading frame. Cleavage by TEV protease liberates GFP with an N- terminal serine, which is stable according to the N-end rule. D. Western analysis of the mcm7-GFP_N760::Tcs strains, using anti-GFP antibody. Lane 1: untagged control strain (P138); lane 2: mcm7-GFP_N760::Tcs strain (P1288); lane 3: mcm7-GFP_N760::Tcs containing pREP3X-TEV-NLS, +thiamine (P1171); lane 4: mcm7-GFP_N760::Tcs strain containing pREP3X-TEV-NLS, -thiamine (P1171). All cultures were grown at 32°C. D. Fluorescence microscopy analysis of cells expressing Mcm7-GFP_N760::Tcs in the absence (+thiamine) or presence (-thiamine) of TEV protease. Cells were fixed using methanol and acetone.

Figure 2

Construction and characterization of a strain expressing Cdc23-CFP with an internal TEV cleavage site (Cdc23_S424::Tcs). A. Regions of Cdc23 with attributed functions (see text for details), and location of Tcs in Cdc23_S424::Tcs. The grey bar superimposed on the black Zn-finger region corresponds to the domain of S. cerevisiae Mcm10 required for homocomplex assembly [31]. "ts" indicates the location of cdc23-1E2 and -M36 ts mutations. B. Expression of TEV protease causes loss of viability of a cdc23_S424::Tcs strain (P1323) at 37°C. Serial dilutions of the P1323 strain were spotted onto minus or plus thiamine media and incubated at the temperatures shown. C. Western analysis of Cdc23 levels in cdc23_S424::Tcs strains in presence or absence of pTEV (pREP3X-TEV-NLS) plasmid, grown in the presence or absence of thiamine. The blot was probed with antibodies against Cdc23, and α-tubulin is shown as a loading control. Cells were first grown at 25°C in the presence of thiamine to repress TEV protease expression, then washed and grown either in the presence or absence of thiamine for 24 h at the indicated temperatures, before extracting protein for western analysis. Cdc23_S424::Tcs indicates the position of full length protein, N & C Cdc23 indicate the fragments (49 kDa (N) and 46 kDa (C)) after cleavage by TEV protease. Comparison of Cdc23 levels in strains 3 and 4 (wt) shows that the modification of Cdc23 does not affect protein levels under conditions where TEV protease is absent. Strains used were: (1) P1323-1; (2) P1323-2; (3); P1322 (4) P138.

Figure 3

Flow cytometry analysis of cdc23_S424::Tcs strains following induction of TEV protease. A. Scheme for experiment shown in (B) & (C). B-C. Flow cytometric analysis of G1-arrested cdc23_S424::Tcs strain (P1376), released from a G1 block in the absence (B) or presence (C) of thiamine, showing arrest of DNA replication when TEV protease is expressed. D. Flow cytometric analysis of cdc23_S424::Tcs strain (P1376) expressing TEV protease (i.e. – thiamine). The strain was grown to log phase at 25°C in – thiamine medium and shifted to 37°C at t = 0. The top panel superimposes the t = 0 (grey) and t = 3 h (black) histograms.

Figure 4

The C-terminal fragment of Cdc23_S424::Tcs is not localized to the nucleus after cleavage by TEV protease. A. cdc23_S424::Tcs strain (P1323) was grown in plus (-TEV) or minus thiamine (+TEV) media at 25°C for 24 h, then shifted to 37°C for 4 h before fixation with methanol and acetone. Panels show Cdc23-CFP fluorescence and DAPI staining superimposed on phase images. Bar = 10 μm. B. Quantitation of experiment shown in A. 100 cells were counted for each time point.

Figure 5

Analysis of Spp1-GFP nuclear distribution after TEV protease cleavage of Cdc23_S424::Tcs. A. Localization of Spp1-GFP after direct fixation (methanol/acetone) of an asynchronous culture (strain P903). B. Localization of Spp1-GFP (strain P903) after detergent extraction of cells; >50% binuclear cells show nuclear Spp1 (arrow) C. As in (B) except that cells were grown in 12 mM HU for 2 h before analysis. Bar = 10 μm. D. Quantitation of data in (B); 100 cells were counted for each data point and 100% represents the total number of binucleate or uninucleate cells counted. E. Quantitation of data in (C); data for other time points (with and without detergent extraction) are also shown. F. Chromatin binding analysis of Spp1-GFP after inactivation of Cdc23_S424::Tcs by TEV protease cleavage. Strain P1460 was grown either in the absence or presence of thiamine for 24 h at 25°C, then shifted to 37°C for 2, 4 h before analysis using the chromatin binding assay. Grey bars show the percentage of total cells showing Spp1-GFP fluorescence coincident with chromatin (DAPI-staining region) after detergent extraction. Black bars show the percentage of cells showing foci of Spp1-GFP fluorescence in the absence of general nuclear fluorescence (example shown in right-hand panels). At least 300 cells were counted for each data point. G. Chromatin association of Cdc45-YFP is not prevented by cleavage of Cdc23_S424::Tcs by TEV protease. Strain P1409 was grown either in the absence or presence of thiamine for 24 h at 25°C, then shifted to 37°C for 2, 4 h before analysis using the chromatin binding assay. Data points show the percentage of total cells showing nuclear retention of Cdc45-YFP after detergent extraction. At all time points, >95% of cells show nuclear Cdc45-YFP before detergent extraction.

Figure 6

Localization of Spp1-GFP and Psf2-YFP after inactivation of Cdc23 using a degron mutant. A. Strain P1205 containing Spp1-GFP in the background of a degron cdc23tstd allele [25] was initially grown at 25°C in the absence of thiamine. 3 h before the temperature shift to 37°C, thiamine was added to repress cdc23 transcription, and 2 h after the shift, cells were analyzed using the chromatin binding assay (focus of Spp1-GFP shown (arrow), bar = 10 μm.) Quantitative analysis is shown in (D). B. Analysis of wild-type Psf2-YFP strain (P1411) in log phase after detergent extraction, showing retention of Psf2 in >50% binucleate (G1 or S phase) cells but not uninucleate (G2) cells. Quantitative analysis is shown in (E). C. Strain P1453, containing Psf2-YFP in the background of a degron cdc23tstd mutant, was treated as in (A) and analyzed by the chromatin binding assay. Cells shown had been shifted to 37°C for 2 h to inactivate Cdc23 before processing using the chromatin binding assay. Quantitative analysis is shown in (E). D. Analysis of data in experiments shown in (A); at least 200 cells were counted for each data point in each experiment. White bars show the percentage of total cells showing Spp1-GFP fluorescence coincident with chromatin (DAPI-staining region) after detergent extraction. Black bars show the percentage of cells showing foci of Spp1-GFP fluorescence in the absence of general nuclear fluorescence. 100% represents total number of cells counted for each data point. E. Analysis of Psf2-YFP in nuclei of wild-type cells, and in a cdc23tstd degron mutant at permissive and non-permissive temperatures, either fixed directly (-detergent), or after detergent extraction (+detergent). For the experiment with the cdc23tstd mutant, the percentages of all cells (i.e. binucleate and uninucleate) are shown.