Drosophila replication and repair proteins: proliferating cell nuclear antigen (PCNA).

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported. Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli. An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity. Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos. This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair.

Architecture of the active DNA polymerase delta.proliferating cell nuclear antigen.template-primer complex.

The relative positions of components of the DNA-dependent DNA polymerase delta (pol delta).proliferating cell nuclear antigen (PCNA).DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol delta was nearly totally (95%) inhibited by a biotin. streptavidin complex located at the 3'-end of a template with a 15-mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin. streptavidin complex. Quantification of results with primers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol delta, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide. Interaction apparently takes place through the C terminus of p125. Based on these results, we conclude that PCNA is located "behind" pol delta in the polymerization complex during DNA synthesis and that only the large subunit of pol delta (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed.

A two-dimensional support for selective binding of polyhistidine-tagged proteins: identification of a proliferating cell nuclear antigen point mutant with altered function in vitro.

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.

Proliferating cell nuclear antigen promotes DNA synthesis past template lesions by mammalian DNA polymerase delta.

Consistent with previous observations, proliferating cell nuclear antigen (PCNA) promotes DNA synthesis by calf thymus DNA polymerase delta (pol delta) past several chemically defined template lesions including model abasic sites, 8-oxo-deoxyguanosine (dG) and aminofluorene-dG (but not acetylaminofluorene-dG). This synthesis is potentially mutagenic. The model abasic site was studied most extensively. When all deoxyribonucleoside triphosphates and a template bearing a model abasic site were present, DNA synthesis by pol delta beyond this site was stimulated 53-fold by addition of homologous PCNA. On an unmodified template (lacking any lesions), PCNA stimulated pol delta by 1.3-fold. Product analysis demonstrated that as expected from the "A-rule," fully and near-fully extended primers incorporated predominantly dAMP opposite the template lesion. Moreover, corollary primer extension studies demonstrated that in the presence (but not the absence) of PCNA, pol delta preferentially elongated primers containing dAMP opposite the model abasic template site. p21, a specific inhibitor of PCNA-dependent DNA replication, inhibits PCNA-stimulated synthesis past model abasic template sites. We propose that DNA synthesis past template lesions by pol delta promoted by PCNA results from the fundamental mechanism by which PCNA stimulates pol delta, i.e., stabilization of the pol delta. template-primer complex.

Proliferating cell nuclear antigen promotes misincorporation catalyzed by calf thymus DNA polymerase delta.

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol delta.template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol delta. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'-exonuclease of pol delta.

The mammalian DNA polymerase delta--proliferating cell nuclear antigen--template-primer complex: molecular characterization by direct binding.

Three direct assays, polyacrylamide gel electrophoresis-band mobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA polymerase delta (pol delta), proliferating cell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol delta, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNase I. The mass determined for the pol delta.PCNA.template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol delta, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol delta.template-primer complex.

Human DNA polymerase epsilon: enzymologic mechanism and gap-filling synthesis.

DNA polymerase epsilon (pol epsilon) was purified to apparent homogeneity from human placentas. The purified enzyme contains a single polypeptide of approximately 170 kDa (apparent mass) and has both DNA polymerase and 3'-5'-exonuclease activities. Competitive inhibition studies indicate that like DNA polymerases alpha and delta (pol alpha and pol delta, respectively), free pol epsilon binds single-stranded but not double-stranded DNA. This conclusion was confirmed by sedimentation binding analysis. Also like pol alpha and pol beta, pol epsilon exhibits induced dNTP inhibition in the presence of template annealed to complementary primer containing a 2',3'-H (dideoxy)-terminus. Together, these data suggest that pol epsilon follows an ordered sequential ter-reactant mechanism of substrate recognition and binding; it binds template first followed by annealed primer and then template-specified dNTP. Enzymologic studies suggest that in contrast to both pol alpha and pol delta, pol epsilon functions more efficiently as gap size decreases. This observation is consistent with a specific role for pol epsilon in gap-filling in vivo. Gap-filling is essential for both replication and repair.