Site-specific mutagenesis of Drosophila proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase σ

Abstract

Background: We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase σ(pol σ). In the presence of homologous PCNA, pol σ exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t1/2 of the pol σ·PCNA·template-primer complex ≥2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes. Results: Drosophila PCNA, although highly similar in structure to mammalian PCNA (e.g., it is >70% identical to human PCNA in amino acid sequence), can only substitute poorly for either calf thymus or human PCNA (∼10% as well) in affecting calf thymus pol σ. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol σ, all four effects can be enhanced dramatically. Conclusions: Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol σ interaction. Moreover unlike mammals, Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol σ for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol σ preparations.