Kathleen M. Downey

Eukaryotic DNA Replication

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.

The benzylthio-pyrimidine U-31,355, a potent inhibitor of HIV-1 reverse transcriptase

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.

Degradation of DNA by 1,-10-phenanthroline.

Summary The metal chelator 1,10-phenanthroline, in the presence of a reducing agent and a copper salt, causes the degradation of double-stranded DNA to acid soluble fragments. The degradation of DNA is dependent on the presence of O2. The concentrations of 1,10-phenanthroline which are effective in degrading DNA are of the same order of magnitude as those that result in inhibition of nucleotidyl transferase reactions. The requirement for a copper salt can only be demonstrated when all reagents are treated with Chelex to remove metal contaminants. It is proposed that the degradation of DNA in the presence of 1,10-phenanthroline may account for the in vitro inhibition of DNA and RNA synthesis seen with this metal chelator, rather than any effect on nucleotidyl transferases.

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.

INCORPORATION AND EXCISION OF 9-(2-PHOSPHONYLMETHOXYETHYL)GUANINE (PMEG) BY DNA POLYMERASE DELTA AND EPSILON IN VITRO

PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its anti-proliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) δ and ε and found it to be a competitive inhibitor of both these enzymes. The apparentK i values for PMEGpp were 3–4 times lower than theK m values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol δ and pol ε to repair the incorporated PMEG revealed that pol ε could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control template-primers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3′-5′-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol ε, pol δ exhibited negligible activity on these template-primers, indicating that pol ε, but not pol δ, can repair the incorporated analog.

Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA polymerase delta processivity.

We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants. Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Δpcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA polymerase δ holoenzyme similar to calf thymus PCNA. Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA polymerase δ but showed no deficiency in stimulation of the ATPase activity of replication factor C. S. pombe Δpcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes. These cells, however, were not sensitive to UV irradiation. Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA polymerase δ for processive DNA synthesis during DNA replication.

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.

Degradation of isolated deoxyribonucleic acid mediated by nitroso-chloramphenicol: Possible role in chloramphenicol-induced aplastic anemia

Reduction of the nitro group of chloramphenicol (CAP) gives rise to more highly reactive intermediates which may in involved in the aplastic anemia associated with CAP use. One such intermediate, nitroso-chloramphenicol (NO-CAP), has been found to be a potent agent for mediating degradation of isolated DNA. In a reaction mixture containing 100 microM NO-CAP, 100 microM CuCl2, and 5 mM NADH, 7 micrograms of Escherichia coli [3H]DNA was completely degraded to acid-soluble fragments in 30 min. Damage to DNA was in the form of single-stranded scissions. The requirement for copper was specific, and copper chelating reagents blocked the degradation. The need for a reducing agent could be met equally well by NADH or NADPH, but not by sulfhydryl reagents such as glutathione, dithiothreitol and 2-mercaptoethanol. Oxygen was also necessary for the NO-CAP mediated DNA damage, with reduced forms of oxygen participating in the reaction. A role for H2O2 was indicated by the inhibition of the degradation seen when catalase was included in the mixture. Hydroxyl radicals are known to be produced in the reaction of H2O2 with certain transition metals. Scavangers of hydroxyl radicals also inhibited strand-scission, suggesting that the radicals may be the primary agents in DNA degradation. The importance of the nitroso moiety of NO-CAP was evidenced by the lack of DNA damage seen when NO-CAP was replaced by CAP under the conditions tested.

The effect of 6-mercaptopurine and its derivatives on mammalian RNA-dependent RNA synthesis.

Summary The effect of 6-mercaptopurine (6-MP) and its nucleoside and nucleotide derivatives on the rate of RNA synthesis with reticulocyte RNA-dependent RNA polymerase has been examined. Of the 6-MP derivatives tested only 6-mercaptopurine ribo-5′-monophosphate (6-MPR-P) significantly stimulates the rate of RNA synthesis with hemoglobin mRNA as a template while 6-MP and 6-MPR have little or no effect. Kinetic studies have demonstrated that 6-MPR-P activates the RNA-dependent RNA polymerase by increasing the Vmax of the enzyme rather than by decreasing the Km values for the substrates. It is suggested that the stimulation of RNA-dependent RNA synthesis by 6-MPR-P may explain the transient development of megaloblastic anemia following 6-MP therapy.