Publications by authors named "Robert L Eoff"

65 Publications

Single and double modified salinomycin analogs target stem-like cells in 2D and 3D breast cancer models.

Biomed Pharmacother 2021 Jun 12;141:111815. Epub 2021 Jun 12.

Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.

Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC range of 1.1 ± 0.1-1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44/CD24 stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.
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http://dx.doi.org/10.1016/j.biopha.2021.111815DOI Listing
June 2021

Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.

NAR Cancer 2021 Jun 9;3(2):zcab014. Epub 2021 Apr 9.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.
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http://dx.doi.org/10.1093/narcan/zcab014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034706PMC
June 2021

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

Nucleic Acids Res 2021 02;49(4):2065-2084

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.
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http://dx.doi.org/10.1093/nar/gkab041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913688PMC
February 2021

A Facile Semisynthesis and Evaluation of Garcinoic Acid and Its Analogs for the Inhibition of Human DNA Polymerase β.

Molecules 2020 Dec 11;25(24). Epub 2020 Dec 11.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Garcinoic acid has been identified as an inhibitor of DNA polymerase β (pol β). However, no structure-activity relationship (SAR) studies of garcinoic acid as a pol β inhibitor have been conducted, in part due to the lack of an efficient synthetic method for this natural product and its analogs. We developed an efficient semi-synthetic method for garcinoic acid and its analogs by starting from natural product δ-tocotrienol. Our preliminary SAR studies provided a valuable insight into future discovery of garcinoic acid-based pol β inhibitors.
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http://dx.doi.org/10.3390/molecules25245847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763917PMC
December 2020

Inhibition of Human DNA Polymerases Eta and Kappa by Indole-Derived Molecules Occurs through Distinct Mechanisms.

ACS Chem Biol 2019 06 22;14(6):1337-1351. Epub 2019 May 22.

Department of Biochemistry and Molecular Biology , University of Arkansas for Medical Sciences , Little Rock , Arkansas 72205-7199 , United States.

Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol κ based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol κ than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol κ by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol η based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol κ and ternary complex formation by hpol η. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC value of IAG-10 to submicromolar values, consistent with inhibition of binary complex formation for hpol κ. Chemical footprinting experiments revealed that IAG-10 binds to a cleft between the finger, little finger, and N-clasp domains on hpol κ and that this likely disrupts the interaction between the N-clasp and the TLS pol core. In cell culture, IAG-10 potentiated the antiproliferative activity and DNA damaging effects of TMZ in hpol κ-proficient cells but not in hpol κ-deficient cells, indicative of a target-dependent effect. Mutagenic replication across alkylation damage increased in hpol κ-proficient cells treated with IAG-10, while no change in mutation frequency was observed for hpol κ-deficient cells. In summary, we developed a potent and selective small-molecule inhibitor of hpol κ that takes advantage of structural features unique to this TLS enzyme to potentiate TMZ, a standard-of-care drug used in the treatment of malignant brain tumors. Furthermore, the IAG scaffold represents a new chemical space for the exploration of TLS pol inhibitors, which could prove useful as a strategy for improving patient response to genotoxic drugs.
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http://dx.doi.org/10.1021/acschembio.9b00304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357868PMC
June 2019

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation.

Nucleic Acids Res 2019 07;47(12):6236-6249

Department of Radiation Oncology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.
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http://dx.doi.org/10.1093/nar/gkz263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614850PMC
July 2019

A Small-Molecule Inhibitor of Human DNA Polymerase η Potentiates the Effects of Cisplatin in Tumor Cells.

Biochemistry 2018 02 30;57(7):1262-1273. Epub 2018 Jan 30.

Department of Biochemistry and Molecular Biology, ‡Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.

Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC value of 8 μM and exhibited 5-10-fold specificity for hpol η over replicative pols. We conclude from kinetic analyses, chemical footprinting assays, and molecular docking that PNR-7-02 binds to a site on the little finger domain and interferes with the proper orientation of template DNA to inhibit hpol η. A synergistic increase in CDDP toxicity was observed in hpol η-proficient cells co-treated with PNR-7-02 (combination index values = 0.4-0.6). Increased γH2AX formation accompanied treatment of hpol η-proficient cells with CDDP and PNR-7-02. Importantly, PNR-7-02 did not impact the effect of CDDP on cell viability or γH2AX in hpol η-deficient cells. In summary, we observed hpol η-dependent effects on DNA damage/replication stress and sensitivity to CDDP in cells treated with PNR-7-02. The ability to employ a small-molecule inhibitor of hpol η to improve the cytotoxic effect of CDDP may aid in the development of more effective chemotherapeutic strategies.
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http://dx.doi.org/10.1021/acs.biochem.7b01176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312786PMC
February 2018

Translesion DNA Synthesis in Cancer: Molecular Mechanisms and Therapeutic Opportunities.

Chem Res Toxicol 2017 11 28;30(11):1942-1955. Epub 2017 Sep 28.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.

The genomic landscape of cancer is one marred by instability, but the mechanisms that underlie these alterations are multifaceted and remain a topic of intense research. Cellular responses to DNA damage and/or replication stress can affect genome stability in tumors and influence the response of patients to therapy. In addition to direct repair, DNA damage tolerance (DDT) is an element of genomic maintenance programs that contributes to the etiology of several types of cancer. DDT mechanisms primarily act to resolve replication stress, and this can influence the effectiveness of genotoxic drugs. Translesion DNA synthesis (TLS) is an important component of DDT that facilitates direct bypass of DNA adducts and other barriers to replication. The central role of TLS in the bypass of drug-induced DNA lesions, the promotion of tumor heterogeneity, and the involvement of these enzymes in the maintenance of the cancer stem cell niche presents an opportunity to leverage inhibition of TLS as a way of improving existing therapies. In the review that follows, we summarize mechanisms of DDT, misregulation of TLS in cancer, and discuss the potential for targeting these pathways as a means of improving cancer therapies.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135728PMC
November 2017

A catch and release program for single-stranded DNA.

J Biol Chem 2017 08;292(31):13085-13086

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205. Electronic address:

Uncovering the mechanisms by which single-stranded binding proteins both protect and expose single-stranded DNA has important implications for our understanding of DNA replication and repair. A new study serves up a master class in developing a full kinetic model for one such protein, mtSSB, showing how DNA can be reeled in and set free to control accessibility.
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http://dx.doi.org/10.1074/jbc.H117.791392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546045PMC
August 2017

Synthesis and Evaluation of 2-Naphthaleno trans-Stilbenes and Cyanostilbenes as Anticancer Agents.

Anticancer Agents Med Chem 2018 ;18(4):556-564

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.

Background: Naphthalene is a good structural replacement for the isovanillin moiety (i.e. the 3- hydroxy-4-methoxyphenyl unit) in the combretastatin A-4 molecule, a natural product structurally related to resveratrol, which consistently led to the generation of highly cytotoxic naphthalene analogues when combined with a 3,4,5-trimethoxyphenyl or related aromatic system. Also, the naphthalene ring system is present in many current drug molecules that are utilized for anti-tumor, anti-arrhythmia and antioxidant therapy.

Objective: In our continuing quest to improve the potencies of naturally occurring anti-cancer molecules through chemical modification, we have now synthesized a small library of 2-naphthaleno trans- stilbenes and cyanostilbenes that are structurally related to both resveratrol and DMU-212, and have evaluated these novel analogs against a panel of 54 human tumor cell lines.

Method: A series of 2-naphthaleno-containing trans-stilbenes 3a-3h (Scheme 1) were synthesized by Wittig reaction of a variety of aromatic substituted benzyl-triphenylphosphonium bromide reactants with 2- naphthaldehyde using n-BuLi as a base in THF. A second series of 2-naphthaleno trans-cyanostilbenes analogs 5a-5h was synthesized by reaction of 2-naphthaldehyde (2; 1 mmol) with an appropriately substituted 2- phenylacrylonitrile 4a-4h; 1 mmol) in 5% sodium methoxide/methanol. The reaction mixture was stirred at room temperature for 2-3 hours and the reaction allowed to go to completion (TLC monitoring), during which time the desired product precipitated out of the solution as a solid. The resulting precipitate was filtered off, washed with water and dried to yield the desired compound in yields ranging from 70-95% (Scheme 2).

Results: The percentage growth inhibition of 54 human cancer cell lines in a primary NCI screen after exposure to compounds 3a, 3d, 5b and 5c was carried out. The results showed that only compounds 5b and 5c met the criteria for subsequent testing to determine growth inhibition values (GI50) in dose-response studies. At 10-5 M concentration, compounds 5b and 5c exhibited cytotoxic activity against leukemia cell lines HL-60(TB) and SR, lung cancer cell line NCI-H522, colon cancer cell lines COLO 205 and HCT-116, CNS-cancer cell line SF-539, melanoma cell line MDA-MB-435, and breast cancer cell line BT-549. The naphthalene trans-stilbene analogue 3a, exhibited significant growth inhibition against only one cell line, melanoma cell line MDA-MB-435 (96 % growth inhibition). Compound 3d was inactive in the 10-5 M single dose screen.

Conclusion: We have synthesized a small set of novel 2-naphthaleno stilbenes and cyanostilbenes and evaluated several of these compounds for their anticancer properties against a panel of 54 human tumor cell lines. The most active analogs, 5b and 5c, showed significantly improved growth inhibition against the human cancer cells in the NCI panel when compared to DMU-212. Of these compounds, analog 5c was found to be the most potent anticancer agent and exhibited significant growth inhibitory effects against COLO 205, CNS SF 539 and melanoma SK-MEL 5 and MDA-MB-435 cell lines with GI50 values ≤ 25 nM. Analog 5b also exhibited GI50 values in the range 25-41 nM against CNS SF 295 and melanoma MDA-MB-435 and UACC-62 cell lines. Compounds 5b and 5c were also cytotoxic towards the MV4-11 leukemia cell line with LD50 value of 450 nM and 200 nM, respectively, and demonstrated >50% inhibition of tubulin polymerization at concentrations below their LD50 values in these cells. In silico docking studies suggest that compounds 5b and 5c bind favorably at the colchicine- binding pocket of the tubulin dimer, indicating that both 5b and 5c may inhibit tubulin polymerization through a mechanism similar to that exhibited by colchicine. Derivative 5c demonstrated more favorable binding based on the docking score and buried surface area, as compared to compound 5b, in agreement with the higher observed potency of 5c against a broader range of tumor cell lines. Based on these results, analog 5c is considered to be a lead compound for further optimization as a clinical candidate for treating a variety of cancers.
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http://dx.doi.org/10.2174/1871521409666170412115703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584397PMC
July 2019

Residues in the RecQ C-terminal Domain of the Human Werner Syndrome Helicase Are Involved in Unwinding G-quadruplex DNA.

J Biol Chem 2017 02 9;292(8):3154-3163. Epub 2017 Jan 9.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199. Electronic address:

The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates. NMR studies identified residues involved specifically in interactions with G4 DNA. Three of the amino acids in the WRN RQC domain that exhibited the largest G4-specific changes in NMR signal were then mutated alone or in combination. Mutating individual residues implicated in G4 binding had a modest effect on WRN binding to DNA, decreasing the preference for G4 substrates by ∼25%. Mutating two G4-interacting residues (T1024G and T1086G) abrogated preferential binding of WRN to G4 DNA. Very modest decreases in G4 DNA-stimulated ATPase activity were observed for the mutant enzymes. Most strikingly, G4 unwinding by WRN was inhibited ∼50% for all three point mutants and >90% for the WRN double mutant (T1024G/T1086G) relative to normal B-form dsDNA substrates. Our work has helped to identify residues in the WRN RQC domain that are involved specifically in the interaction with G4 DNA.
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http://dx.doi.org/10.1074/jbc.M116.767699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336152PMC
February 2017

Antinociceptive effects of the 6-O-sulfate ester of morphine in normal and diabetic rats: Comparative role of mu- and delta-opioid receptors.

Pharmacol Res 2016 11 13;113(Pt A):335-347. Epub 2016 Sep 13.

Departments of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA. Electronic address:

This study determined the antinociceptive effects of morphine and morphine-6-O-sulfate (M6S) in both normal and diabetic rats, and evaluated the comparative role of mu-opioid receptors (mu-ORs) and delta-opioid receptors (delta-ORs) in the antinociceptive action of these opioids. In vitro characterization of mu-OR and delta-OR-mediated signaling by M6S and morphine in stably transfected Chinese hamster ovary (CHO-K1) cells showed that M6S exhibited a 6-fold higher affinity for delta-ORs and modulated G-protein and adenylyl cyclase activity via delta-ORs more potently than morphine. Interestingly, while morphine acted as a full agonist at delta-ORs in both functional assays examined, M6S exhibited either partial or full agonist activity for modulation of G-protein or adenylyl cyclase activity, respectively. Molecular docking studies indicated that M6S but not morphine binds equally well at the ligand binding site of both mu- and delta-ORs. In vivo analgesic effects of M6S and morphine in both normal and streptozotocin-induced diabetic Sprague-Dawley rats utilizing the hot water tail flick latency test showed that M6S produced more potent antinociception than morphine in both normal rats and diabetic rats. This difference in potency was abrogated following antagonism of delta- but not mu- or kappa (kappa-ORs) opioid receptors. During 9days of chronic treatment, tolerance developed to morphine-treated but not to M6S-treated rats. Rats that developed tolerance to morphine still remained responsive to M6S. Collectively, this study demonstrates that M6S is a potent and efficacious mu/delta opioid analgesic with a delayed tolerance profile when compared to morphine in both normal and diabetic rats.

Perspective: This study demonstrates that M6S acts at both mu- and delta-ORs, and adds to the growing evidence that the use of mixed mu/delta opioid agonists in pain treatment may have clinical benefit.
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http://dx.doi.org/10.1016/j.phrs.2016.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107169PMC
November 2016

Human Translesion Polymerase κ Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA.

Biochemistry 2016 09 7;55(37):5218-29. Epub 2016 Sep 7.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.

We have investigated the in vitro properties of human Y-family polymerase κ (hpol κ) on G-quadruplex DNA (G4 DNA). Similar to hpol η, another Y-family member implicated in replication of G4 motifs, hpol κ bound G4 DNA with a 5.7-fold preference over control, non-G4 DNA. Results from pol extension assays are consistent with the notion that G-quadruplexes present a stronger barrier to DNA synthesis by hpol κ than they do to that by hpol η. However, kinetic analysis revealed that hpol κ activity increases considerably when the enzyme is 2-3 nucleotides from the G4 motif, a trend that was reported previously for hpol η, though the increase was less pronounced. The increase in hpol κ activity on G4 DNA was readily observed in the presence of either potassium or sodium but much less so when lithium was used in the buffer. The increased activity 2-3 nucleotides from the G4 motif was accompanied by a decrease in the fidelity of hpol κ when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol κ decreased progressively as the primer was moved closer than 2 nucleotides from the G4 motif when either potassium or sodium was used to stabilize the G-quadruplex. Interestingly, the decrease in catalytic activity at the site of the quadruplex observed in potassium-containing buffer was accompanied by an increase in fidelity on G4 substrates versus control non-G4 substrates. This trend of increased fidelity in copying a tetrad-associated guanine was observed previously for hpol η, but not for the B-family member hpol ε, which exhibited a large decrease in both efficiency and fidelity in the attempt to copy the first guanine in the G4 motif. In summary, hpol κ activity was enhanced relative to those of other Y-family members when the enzyme is 2-3 nucleotides from the G4 motif, but hpol κ appears to be less competent than hpol η at copying tetrad-associated guanines.
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http://dx.doi.org/10.1021/acs.biochem.6b00374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113810PMC
September 2016

Aberrant Kynurenine Signaling Modulates DNA Replication Stress Factors and Promotes Genomic Instability in Gliomas.

Chem Res Toxicol 2016 09 15;29(9):1369-80. Epub 2016 Aug 15.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , 4301 W. Markham Street, Little Rock, Arkansas 72205-7199, United States.

Metabolism of the essential amino acid L-tryptophan (TRP) is implicated in a number of neurological conditions including depression, neurodegenerative diseases, and cancer. The TRP catabolite kynurenine (KYN) has recently emerged as an important neuroactive factor in brain tumor pathogenesis, with additional studies implicating KYN in other types of cancer. Often highlighted as a modulator of the immune response and a contributor to immune escape for malignant tumors, it is well-known that KYN has effects on the production of the coenzyme nicotinamide adenine dinucleotide (NAD(+)), which can have a direct impact on DNA repair, replication, cell division, redox signaling, and mitochondrial function. Additional effects of KYN signaling are imparted through its role as an endogenous agonist for the aryl hydrocarbon receptor (AhR), and it is largely through activation of the AhR that KYN appears to mediate malignant progression in gliomas. We have recently reported on the ability of KYN signaling to modulate expression of human DNA polymerase kappa (hpol κ), a translesion enzyme involved in bypass of bulky DNA lesions and activation of the replication stress response. Given the impact of KYN on NAD(+) production, AhR signaling, and translesion DNA synthesis, it follows that dysregulation of KYN signaling in cancer may promote malignancy through alterations in the level of endogenous DNA damage and replication stress. In this perspective, we discuss the connections between KYN signaling, DNA damage tolerance, and genomic instability, as they relate to cancer.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129620PMC
September 2016

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress.

J Biol Chem 2016 08 1;291(34):18041-57. Epub 2016 Jul 1.

From the Departments of Biochemistry and Molecular Biology and the Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 and

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.
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http://dx.doi.org/10.1074/jbc.M116.718478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016190PMC
August 2016

Synthesis and anti-cancer screening of novel heterocyclic-(2)-1,2,3-triazoles as potential anti-cancer agents.

Medchemcomm 2015 Aug 30;6(8):1535-1543. Epub 2015 Jun 30.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

-Cyanocombretastatin A-4 (-CA-4) analogues have been structurally modified to afford their more stable CA-4-(2)-1,2,3-triazole analogues. Fifteen novel, stable 4-heteroaryl-5-aryl-(2)-1,2,3-triazole CA-4 analogues (, and ) were evaluated for anti-cancer activity against a panel of 60 human cancer cell lines. These analogues displayed potent cytotoxic activity against both hematological and solid tumor cell lines with GI values in the low nanomolar range. The most potent compound, , was a benzothiophen-2-yl analogue that incorporated a 3,4,5-trimethoxyphenyl moiety connected to the (2)-1,2,3-triazole ring system. Compound exhibited GI values of <10 nM against 80% of the cancer cell lines in the panel. Three triazole analogues, , and , showed particularly potent growth inhibition against the triple negative Hs578T breast cancer cell line with GI values of 10.3 nM, 66.5 nM and 20.3 nM, respectively. Molecular docking studies suggest that these compounds bind to the same hydrophobic pocket at the interface of α- and β-tubulin that is occupied by colchicine and -CA-4, and are stabilized by Van der Waals' interactions with surrounding amino acid residues. Compound was found to inhibit tubulin polymerization with an IC value of 1.7 µM. The potent cytotoxicity of these novel compounds and their inhibition of tubulin dynamics make these triazole analogues promising candidates for development as anti-cancer drugs.
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http://dx.doi.org/10.1039/C5MD00219BDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821443PMC
August 2015

Dioxol and dihydrodioxin analogs of 2- and 3-phenylacetonitriles as potent anti-cancer agents with nanomolar activity against a variety of human cancer cells.

Bioorg Med Chem Lett 2016 May 17;26(9):2164-9. Epub 2016 Mar 17.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Electronic address:

A small library of (Z)-2-(benzo[d][1,3]dioxol-5-yl) and (Z)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl analogs of 2- and 3-phenylacetonitriles has been synthesized and evaluated for their anti-cancer activities against a panel of 60 human cancer cell lines. The dihydrodioxin analog 3j and dioxol analogs 5e and 7e exhibited the most potent anti-cancer activity of all the analogs synthesized in this study, with GI50 values of <100 nM against almost all of the cell lines in the human cancer cell panel. Of these three, only compound 3j inhibited tubulin polymerization to any degree in vitro. The binding modes of 3j and the structurally related tubulin-inhibitor DMU-212 were determined by virtual docking studies with tubulin dimer. Compound 3j docked at the colchicine-binding site at the dimer interface of tubulin. The Full-Fitness (FF) score of 3j was observed to be substantially higher than DMU-212, which agrees well with the observed anti-cancer potency (GI50 values). The mechanism by which dioxol analogs 5e and 7e exert their cytotoxic effects remains unknown at this stage, but it is unlikely that they affect tubulin dynamics. Nevertheless, these findings suggest that both dioxol and dihydrodioxin analogs of phenylacrylonitrile may have potential for development as clinical candidates to treat a variety of human cancers.
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http://dx.doi.org/10.1016/j.bmcl.2016.03.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930014PMC
May 2016

Effects of Twelve Germline Missense Variations on DNA Lesion and G-Quadruplex Bypass Activities of Human DNA Polymerase REV1.

Chem Res Toxicol 2016 Mar 4;29(3):367-79. Epub 2016 Mar 4.

Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

The Y-family DNA polymerase REV1 is involved in replicative bypass of damaged DNA and G-quadruplex (G4) DNA. In addition to a scaffolding role in the replicative bypass, REV1 acts in a catalytic role as a deoxycytidyl transferase opposite some replication stall sites, e.g., apurinic/apyrimidinic (AP) sites, N(2)-guanyl lesions, and G4 sites. We characterized the biochemical properties of 12 reported germline missense variants of human REV1, including the N373S variant associated with high risk of cervical cancer, using the recombinant REV1 (residues 330-833) proteins and DNA templates containing a G, AP site, N(2)-CH2(2-naphthyl)G (N(2)-NaphG), or G4. In steady-state kinetic analyses, the F427L, R434Q, M656V, D700N, R704Q, and P831L variants displayed 2- to 8-fold decreases in kcat/Km for dCTP insertion opposite all four templates, compared to that of wild-type, while the N373S, M407L, and N497S showed 2- to 3-fold increases with all four and the former three or two templates, respectively. The F427L, R434Q, M656V, and R704Q variants also had 2- to 3-fold lower binding affinities to DNA substrates containing G, an AP site, and/or N(2)-NaphG than wild-type. Distinctively, the N373S variant had a 3-fold higher binding affinity to G4 DNA than the wild-type, as well as a 2-fold higher catalytic activity opposite the first tetrad G, suggesting a facilitating effect of this variation on replication of G4 DNA sequences in certain human papillomavirus genomes. Our results suggest that the catalytic function of REV1 is moderately or slightly altered by at least nine genetic variations, and the G4 DNA processing function of REV1 is slightly enhanced by the N373S variation, which might provide the possibility that certain germline missense REV1 variations affect the individual susceptibility to carcinogenesis by modifying the capability of REV1 for replicative bypass past DNA lesions and G4 motifs derived from chemical and viral carcinogens.
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http://dx.doi.org/10.1021/acs.chemrestox.5b00513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116390PMC
March 2016

Kynurenine Signaling Increases DNA Polymerase Kappa Expression and Promotes Genomic Instability in Glioblastoma Cells.

Chem Res Toxicol 2016 Jan 30;29(1):101-8. Epub 2015 Dec 30.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.

Overexpression of the translesion synthesis polymerase hpol κ in glioblastomas has been linked to poor patient prognosis; however, the mechanism promoting higher expression in these tumors remains unknown. We determined that activation of the aryl hydrocarbon receptor (AhR) pathway in glioblastoma cells leads to increased hpol κ mRNA and protein levels. We blocked nuclear translocation and DNA binding by AhR in glioblastoma cells using a small-molecule and observed decreased hpol κ expression. Pharmacological inhibition of tryptophan-2,3-dioxygenase (TDO), the enzyme largely responsible for activating AhR in glioblastoma, led to a decrease in the endogenous AhR agonist kynurenine and a corresponding decrease in hpol κ protein levels. Importantly, we discovered that inhibiting TDO activity, AhR signaling, or suppressing hpol κ expression with RNA interference led to decreased chromosomal damage in glioblastoma cells. Epistasis assays further supported the idea that TDO activity, activation of AhR signaling, and the resulting overexpression of hpol κ function primarily in the same pathway to increase endogenous DNA damage. These findings indicate that upregulation of hpol κ through glioblastoma-specific TDO activity and activation of AhR signaling likely contributes to the high levels of replication stress and genomic instability observed in these tumors.
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http://dx.doi.org/10.1021/acs.chemrestox.5b00452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718841PMC
January 2016

1-Benzyl-2-methyl-3-indolylmethylene barbituric acid derivatives: Anti-cancer agents that target nucleophosmin 1 (NPM1).

Bioorg Med Chem 2015 Nov;23(22):7226-33

In the present study, we have designed and synthesized a series of 1-benzyl-2-methyl-3-indolylmethylene barbituric acid analogs (7a-7h) and 1-benzyl-2-methyl-3-indolylmethylene thiobarbituric acid analogs (7 i-7 l) as nucleophosmin 1 (NPM1) inhibitors and have evaluated them for their anti-cancer activity against a panel of 60 different human cancer cell lines. Among these analogs 7 i, 7 j, and 7 k demonstrated potent growth inhibitory effects in various cancer cell types with GI50 values <2 μM. Compound 7 k exhibited growth inhibitory effects on a sub-panel of six leukemia cell lines with GI50 values in the range 0.22-0.35 μM. Analog 7 i also exhibited GI50 values <0.35 μM against three of the leukemia cell lines in the sub-panel. Analogs 7 i, 7 j, 7 k and 7 l were also evaluated against the mutant NPM1 expressing OCI-AML3 cell line and compounds 7 k and 7 l were found to cause dose-dependent apoptosis (AP50 = 1.75 μM and 3.3 μM, respectively). Compound 7k also exhibited potent growth inhibition against a wide variety of solid tumor cell lines: that is, A498 renal cancer (GI50 = 0.19 μM), HOP-92 and NCI-H522 lung cancer (GI50 = 0.25 μM), COLO 205 and HCT-116 colon cancer (GI50 = 0.20 and 0.26 μM, respectively), CNS cancer SF-539 (GI50 = 0.22 μM), melanoma MDA-MB-435 (GI50 = 0.22 μM), and breast cancer HS 578T (GI50 = 0.22 μM) cell lines. Molecular docking studies suggest that compounds 7 k and 7 l exert their anti-leukemic activity by binding to a pocket in the central channel of the NPM1 pentameric structure. These results indicate that the small molecule inhibitors 7 i, 7 j, 7 k, and 7 l could be potentially developed into anti-NPM1 drugs for the treatment of a variety of hematologic malignancies and solid tumors.
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http://dx.doi.org/10.1016/j.bmc.2015.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747820PMC
November 2015

Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles as cis-constrained analogues of combretastatin A-4.

Eur J Med Chem 2015 Oct 29;103:123-32. Epub 2015 Aug 29.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA. Electronic address:

A series of combretastatin A-4 (CA-4) analogues have been prepared from (Z)-substituted diarylacrylonitriles (1a-1p) obtained in a two-step synthesis from appropriate arylaldehydes and acrylonitriles. The resulting 4,5-disubstituted 2H-1,2,3-triazoles were evaluated for their anti-cancer activities against a panel of 60 human cancer cell lines. The diarylacrylonitrile analogue 2l exhibited the most potent anti-cancer activity in the screening studies, with GI₅₀ values of <10 nM against almost all the cell lines in the human cancer cell panel and TGI values of <10 nM against cancer cell lines SF-539, MDA-MB-435, OVCAR-3 and A498. Furthermore, in silico docking studies of compounds 2l, 2e and 2h within the active site of tubulin were carried out in order to rationalize the mechanism of the anti-cancer properties of these compounds. From the in silico studies, compound 2e was predicted to have better affinity for the colchicine binding site on tubulin compared to compounds 2l and 2h. Analogue 2e was also evaluated for its anti-cancer activity by colony formation assay against 9LSF rat gliosarcoma cells and afforded an LD₅₀ of 7.5 nM. A cell cycle redistribution assay using analogue 2e was conducted to further understand the mechanism of action of these CA-4 analogues. From this study, analogues 2e and 2l were the most potent anti-cancer agents in this structural class, and were considered lead compounds for further development as anti-cancer drugs.
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http://dx.doi.org/10.1016/j.ejmech.2015.08.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631050PMC
October 2015

Synthesis and evaluation of a series of resveratrol analogues as potent anti-cancer agents that target tubulin.

Medchemcomm 2015 May;6(3):788-794

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

A series of novel diarylacrylonitrile and -stilbene analogues of resveratrol has been synthesized and evaluated for their anticancer activities against a panel of 60 human cancer cell lines. The diarylacrylonitrile analogues and exhibited the most potent anticancer activity of all the analogues synthesized in this study, with GI values of < 10 nM against almost all the cell lines in the human cancer cell panel. Compounds and were also screened against the acute myeloid leukemia (AML) cell line, MV4-11, and were found to have potent cytotoxic properties that are likely mediated through inhibition of tubulin polymerization. Results from molecular docking studies indicate a common binding site for and on the 3,3-tubulin heterodimer, with a slightly more favorable binding for compared to ; this is consistent with the results from the microtubule assays, which demonstrate that is more potent than in inhibiting tubulin polymerization in MV4-11 cells. Taken together, these data suggest that diarylacrylonitriles and may have potential as antitubulin therapeutics for treatment of both solid and hematological tumors.
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http://dx.doi.org/10.1039/C4MD00478GDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4527554PMC
May 2015

Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

Biochemistry 2015 May 11;54(20):3218-30. Epub 2015 May 11.

†Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, United States.

We have investigated the action of the human DNA polymerase ε (hpol ε) and η (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA over non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with nearly equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain >25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold greater than that of hpol ε when comparing the misinsertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model in which a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication.
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http://dx.doi.org/10.1021/acs.biochem.5b00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682564PMC
May 2015

Synthesis, anticancer activity and molecular docking studies on a series of heterocyclic trans-cyanocombretastatin analogues as antitubulin agents.

Eur J Med Chem 2015 Mar 29;92:212-20. Epub 2014 Dec 29.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA. Electronic address:

A series of heterocyclic combretastatin analogues have been synthesized and evaluated for their anticancer activity against a panel of 60 human cancer cell lines. The most potent compounds were two 3,4,5-trimethoxy phenyl analogues containing either an (Z)-indol-2-yl (8) or (Z)-benzo[b]furan-2-yl (12) moiety; these compounds exhibited GI50 values of <10 nM against 74% and 70%, respectively, of the human cancer cell lines in the 60-cell panel. Compounds 8, and 12 and two previously reported compounds in the same structural class, i.e. 29 and 31, also showed potent anti-leukemic activity against leukemia MV4-11 cell lines with LD50 values = 44 nM, 47 nM, 18 nM, and 180 nM, respectively. From the NCI anti-cancer screening results and the data from the in vitro toxicity screening on cultured AML cells, seven compounds: 8, 12, 21, 23, 25, 29 and 31 were screened for their in vitro inhibitory activity on tubulin polymerization in MV4-11 AML cells; at 50 nM, 8 and 29 inhibited polymerization of tubulin by >50%. The binding modes of the three most active compounds (8, 12 and 29) to tubulin were also investigated utilizing molecular docking studies. All three molecules were observed to bind in the same hydrophobic pocket at the interface of α- and β-tubulin that is occupied by colchicine, and were stabilized by van der Waals' interactions with surrounding tubulin residues. The results from the tubulin polymerization and molecular docking studies indicate that compounds 8 and 29 are the most potent anti-leukemic compounds in this structural class, and are considered lead compounds for further development as anti-leukemic drugs.
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http://dx.doi.org/10.1016/j.ejmech.2014.12.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336621PMC
March 2015

The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Nucleic Acids Res 2014 Oct 7;42(19):12027-40. Epub 2014 Oct 7.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA

Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.
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http://dx.doi.org/10.1093/nar/gku913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231769PMC
October 2014

Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Biochemistry 2014 Oct 10;53(41):6584-94. Epub 2014 Oct 10.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.

Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²⁺ as a cofactor, PrimPol binds primer-template DNA with low affinity K(d,DNA) values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn²⁺ (K(d,DNA) = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn²⁺ compared to Mg²⁺ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg²⁺ is the cofactor compared to Mn²⁺. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn²⁺) to 6-fold (Mg²⁺) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn²⁺, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.
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http://dx.doi.org/10.1021/bi501024uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204878PMC
October 2014

Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

Chem Res Toxicol 2014 Oct 9;27(10):1837-52. Epub 2014 Sep 9.

Division of Pharmacology, Department of Molecular Cell Biology, and ‡Department of Physiology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon, Gyeonggi-do 440-746, Republic of Korea.

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.
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http://dx.doi.org/10.1021/tx5002755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203391PMC
October 2014

Elucidation of kinetic mechanisms of human translesion DNA polymerase κ using tryptophan mutants.

FEBS J 2014 Oct 14;281(19):4394-410. Epub 2014 Aug 14.

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI, USA.

To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3'-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat , suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol ) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair.
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http://dx.doi.org/10.1111/febs.12947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182141PMC
October 2014

Human Rev1 polymerase disrupts G-quadruplex DNA.

Nucleic Acids Res 2014 Mar 23;42(5):3272-85. Epub 2013 Dec 23.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA and Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do 440-746, Republic of Korea.

The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4-15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.
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http://dx.doi.org/10.1093/nar/gkt1314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950705PMC
March 2014

Replication, repair, and translesion polymerase bypass of N⁶-oxopropenyl-2'-deoxyadenosine.

Biochemistry 2013 Dec 15;52(48):8766-76. Epub 2013 Nov 15.

A. B. Hancock Jr. Memorial Laboratory for Cancer Research, †Department of Biochemistry, ‡Department of Chemistry, and §Department of Pharmacology, Center in Molecular Toxicology, Vanderbilt Institute of Chemical Biology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

The oxidative stress products malondialdehyde and base propenal react with DNA bases forming the adduction products 3-(2'-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one (M1dG) and N(6)-(oxypropenyl)-2'-deoxyadenosine (OPdA). M1dG is mutagenic in vivo and miscodes in vitro, but little work has been done on OPdA. To improve our understanding of the effect of OPdA on polymerase activity and mutagenicity, we evaluated the ability of the translesion DNA polymerases hPols η, κ, and ι to bypass OPdA in vitro. hPols η and κ inserted dNTPs opposite the lesion and extended the OPdA-modified primer to the terminus. hPol ι inserted dNTPs opposite OPdA but failed to fully extend the primer. Steady-state kinetic analysis indicated that these polymerases preferentially insert dTTP opposite OPdA, although less efficiently than opposite dA. Minimal incorrect base insertion was observed for all polymerases, and dCTP was the primary mis-insertion event. Examining replicative and repair polymerases revealed little effect of OPdA on the Sulfolobus solfataricus polymerase Dpo1 or the Klenow fragment of Escherichia coli DNA polymerase I. Bacteriophage T7 DNA polymerase displayed a reduced level of OPdA bypass compared to unmodified DNA, and OPdA nearly completely blocked the activity of base excision repair polymerase hPol β. This work demonstrates that bypass of OPdA is generally error-free, modestly decreases the catalytic activity of most polymerases, and blocks hPol β polymerase activity. Although mis-insertion opposite OPdA is relatively weak, the efficiency of bypass may introduce A → G transitions observed in vivo.
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http://dx.doi.org/10.1021/bi401103kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925671PMC
December 2013
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