Effect of pH on the Misincorporation Rate of DNA Polymerase η.

The many known eukaryotic DNA polymerases are classified into four families; A, B, X, and Y. Among them, DNA polymerase η, a Y family polymerase, is a low fidelity enzyme that contributes to translesional synthesis and somatic hypermutation. Although a high mutation frequency is observed in immunoglobulin genes, translesional synthesis occurs with a high accuracy. We determined whether the misincorporation rate of DNA polymerase η varies with ambient conditions. It has been reported that DNA polymerase η is unable to exclude water molecules from the active site. This finding suggests that some ions affect hydrogen bond formation at the active site. We focused on the effect of pH and evaluated the misincorporation rate of deoxyguanosine triphosphate (dGTP) opposite template T by DNA polymerase η at various pH levels with a synthetic template-primer. The misincorporation rate of dGTP by DNA polymerase η drastically increased at pH 8.0-9.0 compared with that at pH 6.5-7.5. Kinetic analysis revealed that the Km value for dGTP on the misincorporation opposite template T was markedly affected by pH. However, this drastic change was not seen with the low fidelity DNA polymerase α.

PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Intracellular small RNA-agarose: preparation and application for the analysis of proteins interacted with small RNA.

Recent study suggests that newly found small RNAs, such as microRNA, aptamer RNA and so on, have the important roles on the eukaryotic cell function, and several intracellular small RNAs are identified. However, the exact role of small RNA including the protein factors interacted with them has not been clarified. To search the novel interaction between the intracellular small RNA and specific proteins, we prepared the intracellular small RNA-agarose derived from the egg extract of African claw frog, Xenopus laevis. Using this agarose, several Xenopus proteins those might interact with small RNA were obtained. Among them, four proteins strongly interacted with small RNA were analyzed by mass spectroscopy. Result indicated that they could be heat shock protein 90, heat shock protein 70, and ATP synthase alpha and beta subunits, respectively. In the present paper, the biological utility of intracellular small RNA-agarose is discussed.

Inhibition of DNA polymerase eta by oxetanocin derivatives.

DNA polymerase eta is recently found as a responsible gene product of xeroderma pigmentosum variant. Differently from other eukaryotic DNA polymerases, such as alpha, beta or gamma, eta polymerase is categorized in Y family. Specific inhibitors for DNA polymerases are useful tools to study the exact role of enzyme in the living cells, however, the inhibitor for DNA polymerase eta has not been developed. We examined the inhibitory effects of several sugar-modified nucleotide analogs on DNA polymerase eta. The arabinonucleotides (araCTP), dideoxynucleotides (ddTTP) and 3'-modified nucleotides (3'-dCTP and 3'-azidothymidine tri-phosphate) did not show any inhibitory effect on DNA polymerase eta. On the other hand, the oxetanocin derivatives those have the oxetane ring as a sugar moiety, OXT-GTP and OXT-ATP, strongly inhibited this polymerase. These results suggest that DNA polymerase eta has a unique recognition mode against the sugar moiety of nucleotide compared with other eukaryotic nucleotide polymerases.

Recognition of oxidized thymine base on the single-stranded DNA by replication protein A.

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.

The pH-dependent mismatch formation by DNA polymerase eta.

To clarify the molecular mechanism of mismatch formation by DNA polymerase eta, the pH-dependency of misincorporation of dNTP was studied with the synthetic template-primer. Incorporation of dNTP formed Watson-Crick type base pair, such as the incorporation of dATP opposite template T, was slightly affected by pH between 6.5 to 9.0. On the other hand, the misincorporation rate of dGTP opposite template T by DNA polymerase eta was drastically increased according to the increasing pH. Kinetical analysis revealed that this change might be due to the change of Km value for dGTP rather than that of Vmax value. This suggests that the affinity of dGTP on DNA polymerase eta during the mismatch formation with template T should be affected by pH.

DNA polymerases as targets of anticancer nucleosides.

DNA polymerase is one of the most important target molecules of antitumor agents, especially for antimetabolite nucleosides that include 1-beta-D-arabinofuranosylcytosine (araC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine). There are several subtypes of mammalian DNA polymerases and their localization and function have been clarified. DNA polymerase alpha, delta and epsilon have been implicated to be responsible for DNA replication, whereas DNA polymerase beta, delta and epsilon have been suggested to work in DNA repair. DNA polymerase gamma is encoded in the nucleus but localizes in the mitochondria, and is responsible for the mitochondrial DNA replication. Recently, several antiviral nucleoside analogs were reported to inhibit DNA polymerase gamma after intracellular phosphorylation and cause severe chronic toxicity. 1-(2-Deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (4'-thio-FAC), an antimetabolite similar to araC and gemcitabine, is recently shown by us to be a very promising agent because of its potent antitumor activity by oral administration to mice. We tested for the inhibitory activities of the triphosphates of 4'-thio-FAC and gemcitabine against DNA polymerase alpha, beta and gamma. The triphosphates of 4'-thio-FAC (4'-thio-FACTP) exhibited the potent inhibitory action against DNA polymerase alpha, whereas it showed moderate inhibition against DNA polymerase beta and little inhibition against DNA polymerase gamma. The triphosphate of gemcitabine (dFdCTP) did not show potent inhibition against these three DNA polymerases. Thus, the effect on ribonucleotide reductase was suggested to be more responsible for the antitumor action of gemcitabine. The differences in the mechanisms of action against DNA polymerases between these drugs and other nucleosides were also discussed.

Regulation of eukaryotic DNA replication by proliferation associated protein, PA2G4, in vitro.

To search a novel factor possibly involved in the regulation of DNA replication for eukaryotic cells, we fractionated the Xenopus egg extract frequently used as a cell free DNA replication system, and obtained a single polypeptide of 50 KDa on SDS-polyacrylamide gel electrophoresis. Data of the amino acid sequence reduced from cloned gene of this protein revealed that this might be a Xenopus homolog of human PA2G4 that was known as a proliferation-associated protein or a cell cycle-regulated protein. Recombinant mouse PA2G4 protein also showed the inhibitory effect on the DNA replication reaction in Xenopus egg extract with the single-stranded DNA template. These results strongly suggest that PA2G4 may have an important role on the regulation of DNA replication in eukaryotic cells.

Utilization efficiency of the oxidized purine nucleotide analogs by DNA polymerase eta.

To elucidate the behavior of DNA polymerase eta against the oxidized purine nucleotides, we determined the utilization efficiency of 2-hydroxy-dATP and 8-hydroxy-dGTP by the recombinant yeast DNA polymerase eta using the primer extension assay with the synthetic oligonucleotide template-primers, and compared those by DNA polymerase alpha. Results indicate that DNA polymerase eta incorporates 2-hydroxy-dATP opposite template G in addition to template T and 8-hydroxy-dGTP opposite A in addition to C, respectively. Kinetic analysis revealed that the rate of mutation caused by 2-OH-dATP and 8-OH-dGTP with DNA polymerase eta should be much higher than those with DNA polymerase alpha.

A novel platinum compound inhibits telomerase activity in vitro and reduces telomere length in a human hepatoma cell line.

Telomerase activity is detectable in most human tumors but not in most normal somatic cells or tissues. Telomerase inhibition has, therefore, been proposed as a novel and potentially selective strategy for antitumor therapy. In the present study, we found that platinum compounds, including cisplatin [cis-diamminedichloro-platinum (II)], strongly inhibited the activity of partially purified rat telomerase. Among the agents tested, 2,3-dibromosuccinato [2-(methylaminomethyl)pyridine]platinum (II) (compound E) exhibited the strongest inhibition, with an median inhibitory concentration (IC(50)) of 0.8 micro M. The mode of inhibition was noncompetitive with either dNTPs or TS (first) primer, with K(i) values estimated to be 2.3 or 3.9 micro M for varied TS primer or dNTPs, respectively. Notably, cisplatin also inhibited the telomerase activity, with an IC(50) of 2.0 micro M. Again, the mode of inhibition was noncompetitive, with K(i) values estimated as 7.3 or 8.1 micro M. Preincubation of TS primer with compound E did not affect the telomerase inhibition, whereas preincubation with cisplatin caused remarkable enhancement. Treatment of a human hepatoma cell line HepG2 with a low concentration of compound E gradually reduced the telomere length, indicating that this compound was able to inhibit telomerase in living cells as well as in vitro.

Reactivation of lytic replication from B cells latently infected with Epstein-Barr virus occurs with high S-phase cyclin-dependent kinase activity while inhibiting cellular DNA replication.

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G(0)/G(1) by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21(WAF-1/CIP-1) and p27(KIP-1), followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21(WAF-1/CIP-1), and p27(KIP-1) were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G(1)/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G(1) to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

Novel phenalenone derivatives from a marine-derived fungus exhibit distinct inhibition spectra against eukaryotic DNA polymerases.

A number of compounds used for cancer chemotherapy exert their effects by inhibiting DNA replication. New inhibitors of DNA polymerases, therefore, could be potential candidates for new anti-cancer drugs. This study tested the effects of two phenalenone-skeleton-based compounds, which were isolated from a marine-derived fungus Penicillium sp., sculezonone-B (SCUL-B) and sculezonone-A (SCUL-A), upon DNA polymerase activity. Both compounds inhibited bovine DNA polymerases alpha and gamma, moderately affected the activity of DNA polymerase epsilon, and had almost no effect on HIV-reverse transcriptase and an E. coli DNA polymerase I Klenow fragment. Most notably, whereas SCUL-A inhibited pol beta (IC(50) = 17 microM), SCUL-B has only a weak influence upon this polymerase (IC(50) = 90 microM). Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by either SCUL-A or SCUL-B was competitive with respect to dTTP substrate and noncompetitive with the template-primer. Whereas pol alpha inhibition by SCUL-B is competitive with respect to dATP, the inhibition by SCUL-A was found to be a mixed type with dATP substrate. The K(i) values of SCUL-B were calculated to be 1.8 and 6.8 microM for DNA polymerases alpha and gamma, respectively. The K(i) of DNA polymerase beta for SCUL-A was 12 microM and that for DNA polymerase alpha, 16 microM. Therefore, deletion of the OH-group at C12 enhanced inhibition of DNA polymerase beta. Since computational analyses of these two inhibitors revealed a remarkable difference in the distribution of negative electrostatic charge on the surface of molecules, we infer that different electrostatic charges might elicit different inhibition spectra from these two compounds.

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates.

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.

Kinetic analysis of nucleotides incorporated opposite oxidized thymine bases on template DNA.

Incorporation of nucleotides opposite the oxidized thymine bases on the template by eukaryotic DNA polymerases alpha and gamma was studied using the synthetic oligonucleotides containing 5-formyluracil or 5-hydroxymethyluracil. Primer extension assay revealed that both polymerases could bypass the oxidized thymine lesion, and incorporated dATP and dGTP opposite 5-formyluracil on the template. On the other hand, dATP was an only nucleotide that was incorporated opposite 5-hydroxymethyluracil by both polymerases as far as examined. These results indicate that 5-formyluracil but not 5-hydroxymethyluracil on template DNA may induce the transition mutation of A:T to G:C in eukaryotes.