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author:("papova, Olga")
1.  Kinetic analysis of the unique error signature of human DNA polymerase ν† 
Biochemistry  2011;50(46):10126-10135.
The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage and mitochondrial family members to very low for certain eukaryotic homologues. The latter include Pol ν which, among all A-family polymerases, is uniquely prone to misincorporate dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν’s more accurate Family A homologue, the Klenow fragment of E. coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, kpol. In the sequence context where fidelity is highest, kpol for correct G-dCTP incorporation by Pol ν is ~ 15-fold faster than kpol for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, kpol is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous, and that the relaxed active-site specificity towards the G-dTTP mispair may be associated with its cellular function(s).
doi:10.1021/bi201197p
PMCID: PMC3235957  PMID: 22008035
2.  Biospecimen Reporting for Improved Study Quality (BRISQ) 
Journal of proteome research  2011;10(8):3429-3438.
Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.
doi:10.1021/pr200021n
PMCID: PMC3169291  PMID: 21574648
3.  Biospecimen Reporting for Improved Study Quality 
Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The Biospecimen Reporting for Improved Study Quality guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.
doi:10.1089/bio.2010.0036
PMCID: PMC3142856  PMID: 21826252
4.  Molecular signature and pathway analysis of human primary squamous and adenocarcinoma lung cancers 
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, with a poor response to chemotherapy and low survival rate. This unfavorable treatment response is likely to derive from both late diagnosis and from complex, incompletely understood biology, and heterogeneity among NSCLC subtypes. To define the relative contributions of major cellular pathways to the biogenesis of NSCLC and highlight major differences between NSCLC subtypes, we studied the molecular signatures of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), based on analysis of gene expression and comparison of tumor samples with normal lung tissue. Our results suggest the existence of specific molecular networks and subtype-specific differences between lung ADC and SCC subtypes, mostly found in cell cycle, DNA repair, and metabolic pathways. However, we also observed similarities across major gene interaction networks and pathways in ADC and SCC. These data provide a new insight into the biology of ADC and SCC and can be used to explore novel therapeutic interventions in lung cancer chemoprevention and treatment.
PMCID: PMC3238469  PMID: 22206048
NSCLC; adenocarcinoma; squamous cell carcinoma; molecular signature; gene expression; pathway
5.  Exploring Molecular Pathways of Triple-Negative Breast Cancer 
Genes & Cancer  2011;2(9):870-879.
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with a high rate of proliferation and metastasis, as well as poor prognosis for advanced-stage disease. Although TNBC was previously classified together with basal-like and BRCA1/2-related breast cancers, genomic profiling now shows that there is incomplete overlap, with important distinctions associated with each subtype. The biology of TNBC is still poorly understood; therefore, to define the relative contributions of major cellular pathways in TNBC, we have studied its molecular signature based on analysis of gene expression. Comparisons were then made with normal breast tissue. Our results suggest the existence of molecular networks in TNBC, characterized by explicit alterations in the cell cycle, DNA repair, nucleotide synthesis, metabolic pathways, NF-κB signaling, inflammatory response, and angiogenesis. Moreover, we also characterized TNBC as a cancer of mixed phenotypes, suggesting that TNBC extends beyond the basal-like molecular signature and may constitute an independent subtype of breast cancer. The data provide a new insight into the biology of TNBC.
doi:10.1177/1947601911432496
PMCID: PMC3352156  PMID: 22593799
triple-negative breast cancer (TNBC); molecular pathways; DNA repair; cell cycle; NF-κB; tumor metabolism
6.  DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds 
Biochemistry  2006;45(3):890-898.
We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair in order to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA Polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen bonding ability in the nascent pair, the efficiency (kpol/Kd) of the polymerase reaction is decreased by 30-fold, affecting ground state (Kd) and transition state (kpol) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. By contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions, and more dependent on hydrogen bonding between base-paired partners.
doi:10.1021/bi051792i
PMCID: PMC2567902  PMID: 16411765
7.  Jun NH2-Terminal Kinase Phosphorylation of p53 on Thr-81 Is Important for p53 Stabilization and Transcriptional Activities in Response to Stress 
Molecular and Cellular Biology  2001;21(8):2743-2754.
The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.
doi:10.1128/MCB.21.8.2743-2754.2001
PMCID: PMC86905  PMID: 11283254
8.  Inhibition of c-Jun N-Terminal Kinase 2 Expression Suppresses Growth and Induces Apoptosis of Human Tumor Cells in a p53-Dependent Manner 
Molecular and Cellular Biology  2000;20(5):1713-1722.
c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53−/−, which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21Cip1/Waf1 in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21Cip1/Waf1 expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21Cip1/Waf1 deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21Cip1/Waf1 are important in counteracting these detrimental effects and promoting cell survival.
PMCID: PMC85354  PMID: 10669748
9.  The Jun Kinase 2 Isoform Is Preferentially Required for Epidermal Growth Factor-Induced Transformation of Human A549 Lung Carcinoma Cells 
Molecular and Cellular Biology  1999;19(3):1938-1949.
We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathway in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Bost, R. McKay, N. Dean, and D. Mercola, J. Biol. Chem. 272:33422–33429, 1997). Several isoforms families of JNK, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative splicing of three different genes and have distinct substrate binding properties. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been screened, and two high-affinity and specific candidates have been identified. Antisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 μM but did not alter JNK2 mRNA or protein levels. Conversely, antisense JNK2 specifically eliminated JNK2 steady-state mRNA and protein expression with an EC50 of 0.1 μM. Antisense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-induced total JNK activity, whereas sense and scrambled-sequence control oligonucleotides had no effect. The elimination of mRNA, protein, and JNK activities lasted 48 and 72 h following a single Lipofectin treatment with antisense JNK1 and JNK2, respectively, indicating sufficient duration for examining the impact of specific elimination on the phenotype. Direct proliferation assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did. EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment but not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but not JNK1 is utilized for mediating the effects of EGF. This study represents the first demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.
PMCID: PMC83987  PMID: 10022881
10.  Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion 
Nature Communications  2013;4:2131.
The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides. Here, by using an intramolecular single-molecule FRET assay, the authors establish and characterize the partially closed conformation as a crucial fidelity checkpoint.
doi:10.1038/ncomms3131
PMCID: PMC3715850  PMID: 23831915

Results 1-10 (10)