Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA - Archive ouverte HAL Access content directly
Journal Articles Nucleic Acids Research Year : 1998

Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA

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1
K Piard
  • Function : Author
G Baldacci
  • Function : Author
I Tratner

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.

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hal-03028936 , version 1 (27-11-2020)

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K Piard, G Baldacci, I Tratner. Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA. Nucleic Acids Research, 1998, 26 (11), pp.2598-2605. ⟨10.1093/nar/26.11.2598⟩. ⟨hal-03028936⟩

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