The expression of Pol5 NTD and CTD in trans

We wanted to assay if the NTD and CTD correspond to different functional domains (trans-complementation assay). To do so, we used plasmids containing POL5, or the three longest truncation mutants of Pol5 (ΔN677 or CTD; ΔN658 or CTD+19 aa; Δ679L or NTD; Figure 51A and 51B), C-terminal truncation of 40 amino acids of the NTD (Δ639H, lacking α-helices 29 and 30) (Figure 51B), or N-terminal truncation of 50 amino acids of the CTD (ΔN727, lacking α-helix 31 at the boundary to the NTD and about one third of the disorganized loop) (see Figure 49 and Figure 51B). POL5 genes were fused either to FLAG or GFP tags, as indicated, for further expression analysis by western blotting (Figure 51C). Plasmids were co-transformed in yeast cells in all possible combinations, which produced the presence of at least one NTD and one CTD. In addition, we used an empty vector in combination with the six plasmids as the negative control of the assay (see 4.2.2.7) (Figure 51C).

As in previous assays, growth on galactose containing medium (SCG) indicated equal number of cells spotted for each strain. In addition, it showed the absence of any dominant negative effect induced by the expression of any variant of Pol5 together with the genomic Pol5-WT. As expected, the co-transformation of an empty vector together with a plasmid expressing a functional Pol5-WT protein did not affect yeast growth on glucose containing medium (SCD). Moreover, consistent with the results presented in section 2.4.2.1, none of the yeast strains co-transformed with Pol5 mutants and empty vectors were able to grow upon depletion conditions. Interestingly, both combinations of plasmids co-expressing NTD (Δ679L) and CTD (ΔN677) produced viable yeast cells growing like wildtype cells. Nevertheless, extension of 19 amino acids in the CTD (ΔN658) had a negative effect and it did not rescue cell growth when expressed in combination with the NTD (Δ679L). Accordingly, shortening of the NTD by 40 amino acids from the C-terminal side (Δ639H) did not rescue growth independently of its expression with the extended CTD (ΔN658) or the CTD alone (ΔN677). Even the N-terminal truncation of the CTD by one α-helix and part of the disorganized loop region (ΔN727), which is not essential, did not rescue growth when expressed in combination with the NTD (Δ679L) (Figure 51B).

Figure 51 (previous page): Analysis of trans-complementation and expression levels of selected Pol5 truncation mutants.

(A) Schematic representation of the tertiary structures of Pol5-WT and N- and C-terminal truncation mutants expressed in the trans-complementation assay (B) or analyzed by western blotting (C). NTD or parts of it are depicted in red and CTD is depicted in blue according to the color-code introduced in Figure 47. Names are indicated. (B) Above, schematic representation of primary structures of Pol5 mutant combinations analyzed by trans-complementation assay. Below, trans-complementation test by drop assay to investigate growth of yeast strains co-transformed with plasmids containing the indicated POL5 allele. The tags of the plasmids are given on the left. Growth was tested on galactose and glucose containing minimal media (SCG and SCD) at 30°C by spotting cell concentrations from OD600 = 10-1 (-1) to 10-4 (-4).

Plasmid combinations that rescue growth are written in bold with gray background and are marked with a green hook. Red cross symbolizes inhibited growth. (C) 5 AUs of cells expressing the different Pol5 mutants fused to FLAG or GFP tag (indicated above) were subjected to denaturing protein extraction. 20% of extracts were resolved in 10% SDS-PAGE followed by western blotting. Detection was performed with antibodies against FLAG and GFP for visualization of the respectively tagged Pol5 mutants. The image of the complete western blot analysis is depicted in supplemental Figure 60.

These results suggest that expression of NTD and CTD as independent polypeptides complements all essential functions of Pol5. Moreover, the absence of trans- complementation when the CTD was extended by one α-helix or shortening of the NTD or the CTD at not essential elements might indicate the structural reconstitution of Pol5. The cell extracts of the yeast strains analyzed in trans-complementation were obtained by denaturing protein extraction (see 4.2.2.10) and analyzed by western blotting (see 4.2.5.4). Since Pol5 or its mutant versions were either fused to FLAG or GFP tag, immunodetection was performed with antibodies against FLAG and GFP (see Table 7 and 4.2.5.6) (Figure 51C).

Compared to the protein expression levels of FLAG-Pol5, even higher levels were detected for FLAG-ΔN677 (CTD) independent of the additional expression of GFP-Δ679L (NTD). Expression levels of FLAG-Δ639H and FLAG-ΔN727 were comparable to FLAG-Pol5 levels, whereas expression levels of FLAG-ΔN658 (CTD+19 aa) were strongly reduced in comparison to FLAG-ΔN677 (CTD) levels and they were also significantly lower than FLAG-Pol5 levels. The protein levels detected for FLAG-Δ679L (NTD) were reduced compared to FLAG-ΔN658 but also compared to FLAG-Δ639H (containing its further truncation at the C-terminal region). Furthermore, the levels did not change upon co- expression of GFP-ΔN677 (CTD). Regarding the GFP-tagged proteins, the tendencies of the expression levels comparing the different versions of Pol5 were similar as observed for the FLAG-tagged proteins. The highest expression levels were observed for GFP-ΔN677 (CTD), next for GFP-Pol5 and GFP-ΔN658, and the lowest levels were observed for GFP- Δ679L.

The sizes of the protein bands prove that the co-transformation of both mutant plasmids into one yeast strain did not lead to a recombination event reproducing the wildtype sequence and thus the wildtype Pol5 on one polypeptide chain. Moreover, the stability of NTDs and CTDs of Pol5 did not increase when co-expressed in the same yeast strain.