Jang-Su Park

Zinc Finger of Replication Protein A, a Non-DNA Binding Element, Regulates Its DNA Binding Activity through Redox

Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X 3CX 2–4CX 12–15CX 2C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J.337, 311–317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453–1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124–15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 m NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.

Cytotoxicity of psammaplin A from a two-sponge association may correlate with the inhibition of DNA replication

BackgroundSV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied.MethodsCell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase α-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA).ResultsWe have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase α-primase is one of its main targets.ConclusionTaken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.

Involvement of DNA-dependent Protein Kinase in UV-induced Replication Arrest

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G1/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs+)), as measured by [3H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs−) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs+ cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs− cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs+cells but not in DNA-PKcs− cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs+ cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs− cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.

Two distinct disulfide bonds formed in human heat shock transcription factor 1 act in opposition to regulate its DNA binding activity.

Under circumstances of heat stress, heat shock transcription factor 1 (HSF1) plays important roles in heat shock protein expression. In this study, an increasing concentration of dithiothreitol (DTT) was found to either enhance or inhibit the heat-induced trimerization of HSF1, suggesting the involvement of dual redox-dependent HSF1 activation mechanisms. Our in vitro experiments show that the heat-induced bonding between the cysteine C36 and C103 residues of HSF1 forms an intermolecular disulfide covalent bond (SS-I bond) and that it directly causes HSF1 to trimerize and bond to DNA. Gel filtration assays show that HSF1 can form intermolecular hydrophobic interaction-mediated (iHI-m) noncovalent oligomers. However, the lack of a trimerization domain prevents HSF1 activation, which suggests that iHI-m noncovalent trimerization is a precondition of SS-I bond formation. On the other hand, intramolecular SS-II bond (in which the C153, C373, and C378 residues of HSF1 participate) formation inhibits this iHI-m trimerization, thereby preventing SS-I bond formation and DNA binding. Thus, HSF1 activation is regulated positively by intermolecular SS-I bond formation and negatively by intramolecular SS-II bond formation. Importantly, these two SS bonds confer different DTT sensitivities (the SS-II bond is more sensitive). Therefore, a low concentration of DTT cleaves the SS-II bond but not the SS-I bond and thus improves DNA binding of HSF1, whereas a high concentration DTT cuts both SS bonds and inhibits HSF1 activation. We propose that these interesting effects further explain cellular HSF1 trimerization, DNA binding, and transcription when cells are under stress.

In vitro analysis of the zinc-finger motif in human replication protein A.

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys-->Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPAs function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPAs single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPAs zinc finger domain in its function.

Redox reaction of benzoquinone on a lipid coated glassy carbon electrode

Benzoquinone reduction on a lipid coated glassy carbon electrode has been studied in aqueous solutions employing spectroelectrochemical techniques. The intermediate species, benzoquinone anion radical (BQ + ), thus far believed to be unstable in aqueous solutions, has been shown to be rather stable in a lipid layer on a glassy carbon electrode, especially in a buffered solution. The kinetic parameters D 0 and k 0 for the oxidation reaction of benzoquinone anion radical (BQ + ) on a phosphatidylcholine (PC) coated electrode were determined to be 8.1 X 10 -6 cm 2 s -1 and 3.2 X 10 -2 cm s -1 respectively, by chronocoulometry. The electrochemical data showed that the reduction products of BQ are incorporated into the PC layer. The interaction of the reduction intermediates with PC was investigated in detail with FT-IR and NMR spectroscopy, which showed that the reduction product, the anion radical, was bound to the hydrogen bonded phosphate group in the PC molecule.

Electrochemical and in situ UV-visible spectroscopic behavior of cytochrome c at a cardiolipin-modified electrode

The interaction of cytochrome c (cyt c) with phospholipids was investigated using electrochemical, in-situ UV–visible, and FTIR spectrophotometric methods, which showed that the electrostatic interaction between cyt c and cardiolipin (CL) gave a rapid direct electron transfer. This was studied for the effect of charges of lipids on the redox reaction of cyt c and investigated for electrochemical behavior according to the quantity of CL, the accumulation time, pH, temperature, and the stability of the CL layer. The spectroelectrochemical results showed that only the absorption band appearing at 550 nm, which is one of the Q bands, was directly related to the redox reaction of iron ions in cyt c. The kinetic parameters, DO and ko for the electron transfer reaction of cyt c on the lipid layer were determined to be 3.09 (±0.02)×10−7 and 2.06 (±0.04)×10−3 cm s−1.

Identification of estrogen-like effects and biologically active compounds in river water using bioassays and chemical analysis.

The Nackdong River is the longest river in South Korea and passes through major cities that have several industrial complexes, including chemical, electric, and petrochemical complexes, and municipal characteristics such as apartment complexes. Along the river, the Gumi region has an electric industrial complex and an apartment complex that may be possible point sources of xenoestrogens such as phenolic compounds. To identify the causative chemicals for estrogenic activity in the river water of this region, bioassay-directed chemical analysis was performed. All samples from six sampling sites (an upstream point: S1; hot spot points: S2-1, S2-2, and S2-3; and downstream points: S3, and S4) showed estrogenic activity in the E-screen assay, with bio-EEQs (17beta-E(2)-equivalent quantities) ranging from 25.35-677.15 pg/L. Samples from S2-2, the sampling point downstream of the junction of stream water, and domestic and industrial wastewater, contained the highest estrogenic activity. Since the bio-EEQ of the organic acid fraction (F2) of the S2-2 sample had the highest activity (823.25 pg-EEQ/L) and F2 may contain phenolic compounds, GC-MS analyses for phenolic xenoestrogens were conducted with the organic acid fractions of the river water samples. Six estrogenic phenolic chemicals, 4-NP, BPA, 4-t-OP, 4-t-BP, 4-n-OP, and 4-n-HTP, were detected, with the highest concentrations (I-EEQ) found in S2-2 (231.80 pg/L). Among these phenolic chemicals, 4-NP was the most potent estrogen (bio-EEF; 8.12 x1 0(-5)) and acted as a full agonist. Furthermore, 4-NP was present at levels (2.0 microg/L in S2-2) that can induce VTG induction in fish (>1 microg/L). In addition, we confirmed that river water (S2-2) significantly increased serum VTG levels in crucian carp (Carassius auratus) in a fish exposure experiment under laboratory conditions. Therefore, phenolic xenoestrogens, especially 4-NP, may be the main causative compounds responsible for the estrogenic effect on the Nackdong River.

Influence of divalent metal ions on E2-induced ER pathway in goldfish (Carassius auratus) hepatocytes

Metal ions existing in the environment could influence the estrogen pathway in aquatic animal, but the detailed mechanism is still delusive. We here showed that in male Carassius auratus hepatocytes, copper (Cu) or cadmium (Cd), did not directly induce vitellogenin (VTG) expression. Interestingly, co-exposure with Cd²⁺ (or Cu²⁺) and 17-β-estradiol (E2) greatly increased the VTG level, comparing with single treatment of E2. Meanwhile, Cd²⁺ or Cu²⁺ (but not E2) triggers HSP70 expression. But, mixture of Cd²⁺ or Cu²⁺ with E2 did not obviously raise HSP70 level. E2 also had no obvious effect on reactive oxygen species. Co-treatment of Cd²⁺ and E2 showed no obvious increase compared to single treatment with Cd²⁺. We further assume that Cd²⁺-involved oxidative stress generates misfolded proteins, resulting in the competition of HSP70 proteins from a heterocomplex (with estrogen receptor). Thus, dissociation of the heterocomplex actives the receptor-ligand binding activity and promotes the E2-induced VTG expression.

Aromatic-participant interactions are essential for disulfide-bond-based trimerization in human heat shock transcription factor 1.

Heat shock transcription factor 1 (HSF1) is a central regulator in the heat shock response. However, its trimerization mechanism remains unclear. Here, we demonstrate that three conserved aromatic amino acids (Trp37, Tyr60, and Phe104) are essential for HSF1 trimerization. Point mutation and fluorescence spectroscopy experiments show that an intramolecular interaction between Tyr60 and alpha-helix 1 in the DNA-binding domain stabilizes the HSF1 structure upon heat stress. Furthermore, intermolecular aromatic-aromatic interaction between the Trp37 and Phe104 supports the approach with the Cys36 and Cys103. Thus, the existence of two differential interactions facilitates the formation of intermolecular disulfide bonds, leading to the heat-induced HSF1 trimerization.