title: Observation of multiple isoforms and specific proteolysis patterns of proliferating cell nuclear antigen in the context of cell cycle compartments and sample preparations.
abstract: The proliferating cell nuclear antigen (PCNA) is an essential component for eukaryotic chromosomal DNA replication and repair. PCNA forms a homotrimer ring, which may function as a DNA sliding clamp for DNA polymerases and, possibly, a docking station for other replication- and repair-related proteins. Several reports have suggested the existence of different PCNA isoforms. Here we confirm, using high resolution two-dimensional electrophoresis with narrow pH ranges, the existence of three PCNA isoforms in both Chinese hamster and human breast cancer cells. Among the three isoforms, M or main form is the dominant one throughout the cell cycle while the relative amounts of the minor components A (acidic) and B (basic) forms appear to vary during the cell cycle. We also observed that a specific pattern of PCNA proteolysis occurred during isoelectric focusing in spite of high urea (8 M) and detergent (2% 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate), which was largely inhibited by the proteosome inhibitor MG132 or boiling. Interestingly, the proteolysis pattern was mainly observed with samples isolated from cells in S and G2 phases. A similar but much lower level of PCNA proteolysis also occurred in vivo within the nuclei of the cells in S phase. Taken together, our data are consistent with the idea that the existence of the different isoforms and specific proteolysis of PCNA are relevant to its functions in vivo.
authors.affiliation: Northeastern Ontario Regional Cancer Centre, Ontario, Canada.
authors.forename: Stanislav N
authors.initials: SN
authors.lastname: Naryzhny
authors.forename: Hoyun
authors.initials: H
authors.lastname: Lee
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D001943:Breast Neoplasms / Q000378:metabolism*; D016466:CHO Cells; D002453:Cell Cycle; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005720:Gamma Rays / Q000009:adverse effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D020033:Protein Isoforms