Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis.
BACKGROUND
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.
Kim
Byung Ju
BJ
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 5M8, Canada.
Lee
Hoyun
H
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D053938:DNA Fragmentation; D004260:DNA Repair; D004261:DNA Replication; D006367:HeLa Cells; D006570:Heterochromatin / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport
D006570:Heterochromatin; D019913:Nuclear Localization Signals; D018809:Proliferating Cell Nuclear Antigen; D053148:Caspase 3; D008239:Lysine