Background information: PCNA (proliferating cell nuclear antigen) is required for a wide range of cellular functions, including DNA replication and damage repair. To be functional, PCNA must associate with the replication and repair foci. In addition, PCNA also mediates targeting of certain replication and repair proteins to these foci. However, the mechanism is not yet known by which PCNA is imported into the nucleus, and then localized to the replication and repair foci.
Results: We have found that an NLS (nuclear localization sequence) is present within the amino acid 101-120 segment of PCNA. An NLS-deleted PCNA was localized in the cytoplasm and showed 5-fold lower affinity for importin-beta than wild-type, suggesting that PCNA may be imported into the nucleus by importin-beta via its NLS. We previously reported that the functional unit of PCNA is a double trimer (as opposed to single homotrimer), and Lys-110 is essential for the formation of the double trimer complex [Naryzhny, Zhao and Lee (2005) J. Biol. Chem. 280, 13888-13894]. The present study shows that the substitution of Lys-110 within the NLS to an alanine residue did not affect its nuclear localization. However, the double-trimer-defective PCNA(K110A) was not localized at replication or repair foci. In contrast, the double-trimer-intact PCNA(K117A) mutant was targeted normally to replication and repair foci. Interestingly, in cells transfected with PCNA(K110A), but not PCNA(K117A), caspase-3-mediated chromosome fragmentation was activated.
Conclusions: The present study suggests that the regulation of PCNA is intimately connected with that of DNA replication, repair and cell death signals, and raises the possibility that defects in the formation of the PCNA double-trimer complex can cause apoptosis.