Robert J. Hickey

Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5′-Phosphorylated DNA Double-Strand Breaks by Replication Runoff

ABSTRACT Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3′ DNA ends are extended by DNA polymerase in vivo closely to the 5′ ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5′ ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5′ kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.

Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis.

The recently discovered human enzyme DNA polymerase ι (pol ι) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol ι in DNA lesion bypass, there is no in vivo evidence to confirm this action. We report here that pol ι expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. We also demonstrate that UV treatment of breast cancer cells additionally increases pol ι expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol ι expression is an early event after UV-mediated DNA damage. That pol ι may play a role in the higher mutation frequencies observed in breast cancer cells was suggested when a reduction in mutation frequency was found after pol ι was immunodepleted from nuclear extracts of the cells. Analysis of the UV-induced mutation spectra revealed that >90% were point mutations. The analysis also demonstrated a decreased C→T nucleotide transition and an increased C→A transversion rate. Overall, our data strongly suggest that pol ι may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.

Synthesis and Preliminary Pharmacological Evaluation of New ( ) 1,4-Naphthoquinones Structurally Related to Lapachol

Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated arylmercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphthoquinone nucleus. Pharmacological screening provided evidence of significant biological activities, including effects against proliferation of the MCF-7 human breast cancer cell line, against Herpes Simplex Virus type 2 infection, and against snake poison-induced myotoxicity. One derivative displaced flunitrazepam binding and showed benzodiazepine-like activity, suggesting novel neuroactive structural motifs.

DNMT1 is a component of a multiprotein DNA replication complex.

DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase a activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.

Effects of gemcitabine and araC on in vitro DNA synthesis mediated by the human breast cell DNA synthesome.

Purpose: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. Methods: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects of dFdCTP and araCTP on the synthesome-associated DNA polymerase α and δ activities. Results: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 μM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 μM dCTP, 3 μM dFdCTP and 60 μM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases α and δ. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase δ activity were much higher than those required to inhibit 50% of DNA polymerase α activity for both dFdCTP and araCTP. Conclusion: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase α activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.

Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity.

Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin‐specific large T‐antigen‐dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase α and DNA polymerase δ, respectively.

Ara-C affects formation of cancer cell DNA synthesome replication intermediates

Purpose: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. Methods: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed using neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases α and δ). Results: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 μM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase α enzyme while the DNA polymerase δ seems to be resistant to the inhibitory effect of that drug. Conclusions: Our results indicate that ara-Cs action on DNA replication is mediated primarily through DNA polymerase α and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.

Ara-C differentially affects multiprotein forms of human cell DNA polymerase

Purpose: The antimetabolite 1-β-d-arabinofuranosylcytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia although the precise mechanism by which ara-C induces cytotoxicity remains unclear. Our laboratory has previously isolated from human cells a DNA replication complex, termed the DNA synthesome, which is fully competent to orchestrate, in vitro, all of the reactions required to efficiently and faithfully replicate DNA. Using this system and the active metabolite of ara-C, ara-CTP, we demonstrated that the human DNA synthesome can efficiently incorporate ara-CTP into internucleotide positions of newly replicated DNA in vitro mimicking results obtained using intact cells and isolated nuclei. We then hypothesized that DNA polymerase auxiliary proteins, present within the DNA synthesome, may aid in incorporating this nucleotide analog into DNA. Methods: To test this hypothesis, we utilized three distinct multiprotein complexes each of which contained human DNA polymerase α and examined with standard in vitro polymerase assays the effectiveness of ara-C in inhibiting various aspects of their polymerase function. Results and conclusion: These polymerase-mediated elongation assays, which included ara-CTP- or ara-C-containing primers in the reaction mixture, showed that the rate of DNA elongation in the presence of ara-CTP was significantly enhanced when the DNA polymerase was associated with its auxiliary proteins, and that the elongation resulted in the formation of internucleotide ara-CMP. Nevertheless, the enhanced activities resulting from the association of these auxiliary proteins with polymerase α did not fully account for the remarkable efficiency with which the DNA synthesome incorporated ara-C into internucleotide positions during DNA replication.

Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-β-D-arabinofuranosylcytosine triphosphate (ara-CTP)

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.

A novel in vitro model system for studying the action of ara-C

Abstract The antimetabolite 1-β-D-arabinofuranosylcytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia. While ara-C has been implicated as a potent inhibitor of mammalian cell DNA replication, the specific mechanism by which ara-C kills cells is not known. In this report we describe the development of an in vitro model system to study the molecular mechanism of ara-CMP incorporation into DNA. This model system makes use of a recently described human cell multiprotein DNA replication complex (MRC) that is competent to replicate DNA in vitro. The MRC can successfully incorporate ara-CMP into replicating DNA at internucleotide positions. These results are similar to those described for studies using intact cells. This MRC-driven in vitro replication system may therefore serve as a powerful model for the study of anticancer agents that directly affect human cell DNA synthesis.