The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage.
Bottom Line: We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity.This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway.Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.
Affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.Show MeSH
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Mentions: We sought to further characterize the binding between Mcm10 and Mec3 in the hopes of identifying a separation-of-function mutant of Mcm10 that would retain its ability to interact with PCNA, but would be deficient in Mec3 binding. Toward this end, we constructed N-terminal truncation mutants of Mcm10 that left the PIP box intact, but deleted the first 50, 100 or 150 amino acids (Figure 2A). This region of Mcm10 lies outside the conserved OB-fold/Zn-finger domain and is not predicted to have any significant secondary structure (17), except for a coiled-coil region at the very N-terminus (21). All fusion proteins were expressed (Figure 2B and Supplementary Figure S1) and we then assessed binding to Mec3 via two-hybrid analyses (Figure 2C). Loss of the N-terminal 50 or 100 residues was inconsequential for Mec3 binding, but deletion of the next 50 amino acids resulted in a near complete loss of interaction (Figure 2A–C). A second series of constructs was then designed to further narrow down the Mec3-interaction domain between amino acids 100 and 150 of Mcm10 (Mcm10Δ110, McmΔ120, McmΔ130 and McmΔ140) and each construct's expression was confirmed (Figure 2D). The individually truncated proteins showed intermediate levels of interaction and did not clearly implicate any specific subregion in Mec3 binding (Figure 2E). Because the Mcm10Δ150 protein eliminated Mec3 binding in two-hybrid assays, we attempted to construct a mutant that left at least a portion of the N-terminus intact, as the N-terminal domain is important for Mcm10 oligomerization (17,21). Unfortunately, an internal deletion (ID) of residues 100–150 of Mcm10 failed to fully eliminate the interaction with Mec3 (Mcm10 ID in Figure 3A and B). Resigned to using the Mcm10Δ150 mutant in further experiments, we sought to determine this mutant's ability to bind to wild-type Mcm10. Mcm10 binding to the Mcm10Δ150 mutant was reduced by ∼85%, and is similarly reduced in the Mcm10Δ100 and all Mcm10 N-terminal truncation mutants spanning residues 100–150 (Figure 3C). Importantly, the Mcm10Δ100 and Mcm10Δ150 truncation proteins fully retained the capacity to bind PCNA (Supplementary Figure S2), arguing that the inability of Mcm10Δ150 to recognize Mec3 was not due to a distortion of the PIP box. Taken together, we conclude that the N-terminus of Mcm10 harbors a second Mec3 binding site that resides within amino acids 100–150. However, this binding site does not appear to be unique, but can be substituted by redundant sites in the N-terminus. Thus, the most reliable way for us to disrupt the interaction between Mcm10 and Mec3 was by deleting all redundant sites within the first 150 amino acids. Since this truncation also eliminated Mcm10:Mcm10 self-interaction, we included an mcm10Δ100 mutant into our subsequent genetic analyses to distinguish between phenotypes caused by the loss of self-interaction or the combined loss of self-interaction and Mec3 binding.
Affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.