Tyrosine 114 is essential for the trimeric structure and the functional activities of human proliferating cell nuclear antigen.

Abstract:

In order to study the effect of trimerization of proliferating cell nuclear antigen (PCNA) on its interaction with DNA polymerase (pol) delta and its loading onto DNA by replication factor C (RF-C) we have mutated a single tyrosine residue located at the subunit interface (Tyr114) to alanine. This mutation (Y114A) had a profound effect on PCNA, since it completely abolished trimer formation as seen by glycerol gradient sedimentation and native gel electrophoresis. Furthermore, the mutant protein was unable to stimulate DNA synthesis by pol delta and did not compete effectively with wild-type PCNA for pol delta, although it was able to oligomerize and could to some extent interact with subunits of functionally active PCNA. We thus conclude that PCNA molecules that are not part of a circular trimeric complex cannot interact with the pol delta core. furthermore, the mutant protein could not be loaded onto DNA by RF-C and did not compete with wild-type PCNA for loading onto DNA, indicating that PCNA trimerization may also be a prerequisite for its recognition by RF-C. The adverse effects caused by this single mutation suggest that trimerization of PCNA is essential for the monomers to keep their overall structure and that the structural changes imposed by trimerization are important for interaction with other proteins.

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