Abstract
The formation of a complex between DNA polymerase delta (pol delta) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase delta to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol delta is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol delta and provides evidence for a site on pol delta that is involved in the protein-protein interactions between PCNA and pol delta. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol delta interacts with PCNA.
Highlights
Proliferating cell nuclear antigen (PCNA)1 was originally discovered as an antigen in autoimmune sera from patients with systemic lupus erythematosus and was reported to be found only in actively proliferating cells [1]
A synthetic peptide conforming to the N2 region was found to inhibit PCNA stimulation of pol ␦ isolated from calf thymus [10]. p125 and PCNA co-expressed in Sf9 cells could be co-immunoprecipitated with an antibody to PCNA, showing that the catalytic subunit of DNA polymerase ␦ interacted with PCNA [10]
Co-immunoprecipitation of p125 and PCNA from Crude Calf Thymus and HeLa Extracts and after Their Ectopic Expression in Cultured Cells—Previous studies have demonstrated that human PCNA could be co-immunoprecipitated with the p125 catalytic subunit of pol ␦ from Sf9 insect cell lysates, under conditions where both proteins were overexpressed as recombinant proteins [10]
Summary
Ectopic Expression of p125 and PCNA in COS-7 Cells—COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) containing 10% fetal bovine serum. After three 10-min washes in TBST, the blot was incubated with anti-mouse IgG-horseradish peroxide conjugate diluted in TBST (1:10,000) for 1 h at room temperature with constant rocking. After reaction for 60 min at room temperature, the PCNA was purified on a G25 Sephadex column (5-ml bed volume) equilibrated in phosphate buffered saline containing 1% bovine serum albumin. The nitrocellulose membranes were blocked with 5% nonfat dry milk in TBST for 45 min at room temperature followed by three washes of TBST for 10 min each. The blots were washed five times with TBST for 15 min followed by incubation with streptavidin-horseradish peroxidase conjugate diluted in TBST (1:5000) for 1 h at room temperature with constant rocking. Arbitrary units of activity were calculated as: -galactosidase units ϭ 1000 ϫ A420/(t ϫ V ϫ A600), where t ϭ min of incubation; V ϭ 0.1 ml
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