Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase ␦. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer. mass spectrometry ͉ pathology ͉ posttranslational modification ͉ DNA replication ͉ genome stability

The post-translational modification of proliferating cell nuclear antigen (PCNA) has been implicated in modulating its function for over 20 years. With multiple interacting partners, PCNA is involved in processes ranging from DNA replication and repair to cell cycle control and apoptosis. The ability of PCNA to distinguish between specific binding partners in different tasks is currently of intense interest, and several post-translational modifications have been reported to modulate its function. Unfortunately, these reports have produced contradictory information on the type(s) of modification present on the molecule. Here we report a detailed structural analysis of a single acidic PCNA isoform, cancer-specific polyferating nuclear anitgen (csPCNA), isolated from breast cancer cells by 2D-PAGE and LC-MS/MS. With this approach we fully characterized the csPCNA isoform and confidently identified a single post-translational modification, methyl esterification. Interestingly, the methyl esters consistently localized to 15 specific glutamic and aspartic acid residues of csPCNA. The methyl esterification of csPCNA represents a novel type of post-translational modification in mammalian cells that could ultimately hold the key towards unlocking its diverse functions.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.

Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.