Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast.
The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation.
Das-Bradoo
Sapna
S
University of Minnesota, Biochemistry, Molecular Biology and Biophysics, 321 Church Street SE, 6-155 Jackson Hall, Minneapolis, MN 55455, USA.
Ricke
Robin M
RM
Bielinsky
Anja-Katrin
AK
D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D064110:Minichromosome Maintenance Proteins; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; C076365:MCM10 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D061988:Proteasome Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D064110:Minichromosome Maintenance Proteins