Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA
Open Access
- 1 June 1998
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 26 (11) , 2598-2605
- https://doi.org/10.1093/nar/26.11.2598
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 δ 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 β-sheet βF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.Keywords
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