In this paper we demonstrate that in vivo cleavage by TEV protease can be used to inactivate a modified version of the Cdc23 replication factor, engineered to contain a cleavage site for the protease. We infer that the C-terminal 170 aa region is essential for the DNA replication function of Cdc23. Since N- and C-terminal fragments appear to be stable after cleavage, the C-terminal region must require covalent linkage to the rest of the protein for its function. Although no clear sequence motifs have been identified in the C-terminal region, this part of the protein could have a discrete function that requires linkage to the N-terminal domain. Alternatively, interaction between the N and C-terminal regions could simply be necessary for the function of the protein in a manner which is affected by TEV protease cleavage. This function does not appear to involve nuclear localization or chromatin binding, since nuclear localization of the C-terminal fragment is lost after Cdc23 cleavage (Fig. 4).
We also show that inactivation of Cdc23, either by proteolytic cleavage or using a ts degron allele, affects the chromatin association and nuclear distribution of Spp1 primase subunit (Figs. 5, 6), suggesting that an essential role of Cdc23 is related to recruitment of pol α-primase to chromatin. Cdc45 has also been implicated in binding of pol α-primase to chromatin [37, 39], but in this context it may not be relevant. Although complete inactivation of Cdc23/Mcm10 can block chromatin association of Cdc45 [21, 24, 25], TEV protease cleavage of Cdc23 does not prevent Cdc45 chromatin binding. One interpretation of these results is that Mcm10/Cdc23 has two functions, one related to pol α primase function, perhaps related to the C-terminal domain cleaved off by TEV protease, and the other related to Cdc45 chromatin binding.
These results are consistent with in vitro effects of S. pombe Cdc23 on pol α-primase activity , and evidence that S. cerevisiae Mcm10 promotes chromatin binding of the large primase subunit (Pri2) independently of effects on Cdc45, based on histone cross-linking experiments . The observations we have made for Spp1-GFP may also apply to the three other subunits of pol α-primase (p49/Spp2, p70/B-subunit, p180/Pol1). However, given the requirement for S. cerevisiae Mcm10 for stability of the p180 polymerase catalytic subunit  and the constitutive chromatin binding of at least a fraction of S. pombe p70 , further work will be needed to determine the fate of other pol α primase subunits on Cdc23 inactivation.
In contrast to effects on Spp1, chromatin association of the Psf2 subunit of GINS is seen in cells arrested in S phase by using a degron allele of cdc23 (Fig. 6), similar to the result obtained by an HU arrest of DNA replication. This suggests that maintenance of GINS chromatin association is independent of Cdc23 function and that Psf2 displacement requires Cdc23 function in the elongation step of replication. At first glance, this result is inconsistent with previous work linking Cdc45 with GINS function. Specifically, chromatin association of Sld5 in Xenopus extracts is blocked by Cdc45 depletion , and in S. cerevisiae, the Sld3 partner of Cdc45 is essential for Psf1 origin association . However, degradation of Cdc23 using the cdc23tstd allele used in our experiments is inefficient in cycling cells  and Cdc45 chromatin binding is not blocked under conditions of the Psf2 experiment shown in Fig. 6 (see Additional file: 1).
In this and a related study  we have demonstrated the utility of TEV protease for analysis of protein function in fission yeast. TEV protease can be used to determine the role of specific domains of the protein and this method can be used for topological analysis of protein function by restricting TEV protease expression to specific compartments. For this approach to be useful, the target protein must tolerate the insertion of a Tcs, which was an issue with our related study on Pol3 function  and the Tcs must be accessible to TEV protease in the folded protein. In this study, cleavage of Cdc23S424::Tcs was more efficient at higher temperatures, possibly reflecting a conformational change in Cdc23 which makes the Tcs more accessible to the protease. Alternatively, the interaction of Cdc23 with other factors might be changed by temperature in a manner which affects access to the Tcs.
In the approach used here, the Tcs generates a C-terminal fragment with serine as the N-terminal amino acid, which is predicted to generate a stable protein. However, TEV protease also cleaves sequences such as ENLYFQ∨R [5, 46], which generates a C-terminal fragment with a destabilizing N-terminal amino acid according to the N-end rule. Such a module can be inserted at the N-terminus of the target protein, which may be more tolerated and accessible than insertion of the Tcs at internal sites. We are currently investigating whether this strategy provides a simpler approach to effect rapid inactivation of target proteins.