1787

Proliferating cell nuclear antigen (PCNA) is well known for it ability to support processive DNA synthesis during DNA replication. However, interactions with the repair proteins Msh2 and XPG ascribed roles for PCNA in DNA transactions other than DNA synthesis. Furthermore, after DNA damage, up regulated levels of p21WAF1 has been shown to potently inhibit DNA replication through a direct interaction with PCNA. This interaction was first thought to inhibit DNA replication while allowing DNA repair to occur, but evidence soon emerged suggesting that p21 also inhibited repair. A mechanism as to how PCNA is able to distinguish between its multiple cellular tasks has therefore been elusive. Recently, we have discovered that after treating cells with the DNA damaging agents doxorubicin and cisplatin a transient isoform of PCNA appears. This isoform was termed dPCNA. Analysis of the interaction between p21 and PCNA demonstrated that the association of the two proteins produced dPCNA suggesting that it represents a signaling intermediate during the DNA damage response. To further define dPCNA, cells were pretreated with the inhibitors TSA and anacardic acid prior to treatment with doxorubicin and the ability of the cells to generate dPCNA was examined. Additionally, PCNA was affinity purified from these cells, and upon closer examination multiple different PCNA isoforms were uncovered. LC-MS/MS analysis of these isoforms demonstrated multiple unexpected post-translational modifications to PCNA. Moreover, these sites were partially modified suggesting a heterogeneous population of post-translationally modified species of PCNA present in each spot. Examination of dPCNA by LC-MS/MS demonstrated that it was comprised of PCNA in a distinct post-translational state suggesting that only certain isoforms have dPCNA potential. This data suggests that PCNA is not simply an accessory factor for the DNA polymerases, but is a signaling molecule whose post-translational state determines and directs its cellular function. Inhibition of specific cellular processes by targeting individual PCNA isoforms may therefore represent an attractive new approach towards designing novel therapeutics for the treatment of cancer.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]