Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic function and autoantibody recognition.

Abstract

The proliferating cell nuclear Ag (PCNA) is a DNA replication factor postulated to function as a sliding clamp around DNA. PCNA is also a target for autoimmunity in systemic lupus erythematosus. The autoantigenicity of PCNA is highly conformation-dependent, and reaction with most anti-PCNA sera requires a nearly full-length PCNA molecule. Here we describe the use of gel filtration and glycerol gradient sedimentation to analyze the native structure and size of PCNA. PCNA from three sources was studied (PCNA from HeLa cells, PCNA purified after its overexpression in bacteria, and PCNA produced in the wheat germ cell-free translation system) as well as mutant forms of PCNA translated in vitro. In each case, full-length PCNA behaved as a trimer. Analysis of mutant proteins revealed a correlation between the trimeric form and binding to the common type of human anti-PCNA autoantibody, suggesting that the Abs are specific for the active form of the protein. These findings are consistent with the idea that autoantibodies are generated as a response to native Ag and provide experimental support for the hypothesis that PCNA serves its processive function in DNA replication as a trimeric ring structure.