Characterization of mutant proliferating cell nuclear antigen from aphidicolin-resistant Chinese hamster ovary cells.

摘要

PCNA is a multifunctional protein essential to DNA replication, DNA repair, and cell cycle control. The structure of PCNA has been well characterized; however, there is still much that is unknown about its function and the mechanisms of its interaction. We have chosen to undertake the genetic analysis of PCNA and its interaction with other DNA replication proteins in wild type and mutant cell lines of Chinese Hamster Ovary (CHO) cells resistant to aphidicolin. Previous work from this and other laboratories has demonstrated that the development of aphidicolin resistance is a complex and diverse process involving multiple biochemical alterations, variably including at least one or more of the DNA polymerases.; Here we provide evidence based on cloning, sequence comparison, restriction enzyme analysis, and protein-protein interaction studies that PCNA exists in an altered form in CHO aphidicolin resistant (aphR) cell line BR5 but not in other aphR cell lines. PCNA was cloned from wild type and aphR CHO cells. Sequence analysis of PCNA cloned from aphR cell line Br5, revealed a mutant form of PCNA with nucleotide mutations G/A (254), A/G (546) and T/C (618) in the open reading frame. These nucleotide changes give the inferred amino acid changes A82T, N179S, and V203A. Although none of these mutations are located in the regions of PCNA previously described as functional sites (interdomain connecting loop, center loop, and C-terminal tail), the functional properties of mutant PCNA are clearly altered. The altered properties are dependent on the simultaneous presence of all three mutations. These studies show that mutant PCNA is altered in its binding to GST-cdk2 in vitro. Also, mutant PCNA stimulates the binding of FEN1 to DNA substrate. This is the first evidence of a mutant form of PCNA associated with phenotypically altered cells (aphidicolin resistant) and with functional parameters.