Cheng-Keat Tan

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 δ (pol δ) 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 δ on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol δ 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 δ, the ability to incorporate (and/or not excise) the dTTP analog, 2′-deoxythymidine-5′-O-(α-phosphonomethyl)-β,γ-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol δtemplate-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol δ. This model suggests an explicit mechanistic requirement for the intrinsic 3′-5′-exonuclease of pol δ.

Structural and Functional Properties of Calf Thymus DNA Polymerase δ

Publisher Summary The chapter describes some of the recent studies with DNA polymerase δ from calf thymus. The major topics discussed include (a) evidence that 3‘-to-5’ exonuclease activity is an intrinsic property of DNA polymerase δ; (b) structural properties of the enzyme; (c) evidence that the 3’-to-5’ exonuclease activity has a proof-reading function; and (d) inhibitor studies. The present studies clearly demonstrate that DNA polymerase δ has an associated 3’-to-5’ exonuclease activity. This finding establishes that DNA polymerase δ is a unique enzyme, distinct from other known mammalian DNA polymerases (α, β, γ). The chapter further describes that the 3’-to-5’ exonuclease activity associated with DNA polymerase 6, similar to those of lower eukaryotes and prokaryotes, has a proofreading function. The presence of a proofreading exonuclease in both higher and lower eukaryotic DNA polymerases suggests that the mechanism by which the accuracy of DNA replication is maintained in eukaryotes may be similar to that of prokaryotes. Finally, although inhibitor studies suggest that either DNA polymerase δ or α, or both, may be involved in DNA replication, a biological role for either enzyme in DNA replication remains to be demonstrated.

A tumor necrosis factor α- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase δ and proliferating cell nuclear antigen

A cDNA encoding a protein of 36 kDa, polymerase delta-interacting protein 1 (PDIP1), that interacts with the small subunit (p50) of DNA polymerase δ (pol δ) was identified in a two-hybrid screen of a HepG2 cDNA library by using p50 as bait. The interaction of PDIP1 with p50 was confirmed by pull-down assays, and a similar assay was used to demonstrate that PDIP1 interacts directly with the proliferating cell nuclear antigen (PCNA). PCNA and p50 bound to PDIP1 simultaneously, and PDIP1 stimulated pol δ activity in vitro in the presence, but not the absence, of PCNA, suggesting that PDIP1 also interacts functionally with both p50 and PCNA. Subcellular localization studies demonstrated that PDIP1 is a nuclear protein that colocalizes with PCNA at replication foci. A putative PCNA-binding motif was identified within the C terminus of PDIP1, and a synthetic peptide containing this PCNA-binding motif was shown to bind PCNA by far-Western analysis. Northern analysis demonstrated that PDIP1 mRNA is present in a wide variety of human tissues. PDIP1 was found to be highly homologous to a previously identified protein, B12 [Wolf, F. W., Marks, R. M., Sarma. V., Byers, M. G., Katz, R. W., Shows, T. B. & Dixit, V. M. (1992) J. Biol. Chem. 267, 1317–1326], one of the early response genes induced by tumor necrosis factor α. PDIP1 synthesis can also be induced by tumor necrosis factor α and by IL-6, cytokines essential for liver regeneration after loss of hepatic tissue. It is suggested that PDIP1 provides a link between cytokine activation and DNA replication in liver as well as in other tissues.

PURIFICATION AND CHARACTERIZATION OF THE CATALYTIC SUBUNIT OF HUMAN DNA POLYMERASE DELTA EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS

The catalytic subunit of human DNA polymerase δ has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a Mr = ∼125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase δ. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3′-5′ exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase δ isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn2+ or Mg2+ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn2+ than in Mg2+. The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase δ are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase δ is essential for functional interaction with PCNA.