title: Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA.
abstract: We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene. We thus identified eight lethal mutants out of the 50 cDNAs tested. Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences. Two were full-length with a single point mutation at amino acid 68 or 69. The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified. They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template. Cross-linking experiments showed that this was due to their inability to form trimers. Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.
authors.affiliation: UPR9044, IFC1/CNRS, 7 rue Guy Moquet, 94801 Villejuif, France.
authors.forename: K
authors.initials: K
authors.lastname: Piard
authors.forename: G
authors.initials: G
authors.lastname: Baldacci
authors.forename: I
authors.initials: I
authors.lastname: Tratner
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D015870:Gene Expression; D009687:Nuclear Proteins; D017354:Point Mutation; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D018411:Spodoptera
chemical_list: D005656:Fungal Proteins; D009687:Nuclear Proteins; C078746:PCN1 protein, S pombe; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins