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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.
https://hal-cnrs.archives-ouvertes.fr/hal-03028936
Contributor : Isabelle Tratner Connect in order to contact the contributor
Submitted on : Friday, November 27, 2020 - 6:33:31 PM
Last modification on : Wednesday, January 6, 2021 - 11:34:02 AM
Contributor : Isabelle Tratner Connect in order to contact the contributor
Submitted on : Friday, November 27, 2020 - 6:33:31 PM
Last modification on : Wednesday, January 6, 2021 - 11:34:02 AM
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