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Jerard Hurwitz - One of the best experts on this subject based on the ideXlab platform.
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bypass of a protein barrier by a replicative DNA Helicase
Nature, 2012Co-Authors: Hasan Yardimci, Jerard Hurwitz, Xindan Wang, Anna B Loveland, Inger Tappin, David Z Rudner, Antoine M Van Oijen, Johannes C WalterAbstract:Replicative DNA Helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (“steric exclusion”). In contrast, large T antigen (T-ag), the replicative DNA Helicase of the Simian Virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here, we use singlemolecule and ensemble assays to show that T-ag assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3′ to 5′ direction. Unexpectedly, T-ag unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the T-ag ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative Helicase mechanisms while revealing a new level of plasticity in their interactions with DNA damage.
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selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA Helicase
Cell, 2011Co-Authors: Hasan Yardimci, Jerard Hurwitz, Antoine M Van Oijen, David T Long, Angelo Guainazzi, Vladimir P Bermudez, Orlando D Scharer, Johannes C WalterAbstract:The eukaryotic replicative DNA Helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3′ to 5′ ssDNA translocase, consistent with unwinding via “steric exclusion.” Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.
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processive DNA Helicase activity of the minichromosome maintenance proteins 4 6 and 7 complex requires forked DNA structures
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jerard HurwitzAbstract:The minichromosome maintenance (Mcm) proteins 2–7 are required for both the initiation and elongation steps of chromosomal DNA replication. Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3′ to 5′ DNA Helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt). In this report, we show that the presence of both 5′ and 3′ single-stranded tails in DNA Helicase substrates is essential for the processive Helicase activity of the Mcm complex. The presence of both 5′ and 3′ tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive Helicase activity. The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length. These results support the hypothesis that the Mcm4/6/7 complex can function as a replication Helicase.
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the single minichromosome maintenance protein of methanobacterium thermoautotrophicum δh contains DNA Helicase activity
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Zvi Kelman, Jerard HurwitzAbstract:Previous studies have identified an ATP-dependent DNA Helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508–24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum ΔH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (≈103 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3′-to-5′ ATP-dependent DNA Helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM Helicase activity in eukaryotic chromosomal DNA replication.
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Isolation of Helicase alpha, a DNA Helicase from HeLa cells stimulated by a fork structure and signal-stranded DNA-binding proteins.
The Journal of biological chemistry, 1993Co-Authors: Yeon-soo Seo, Jerard HurwitzAbstract:Abstract A DNA Helicase, called DNA Helicase alpha, was purified from HeLa cells to apparent homogeneity. The Helicase and its single-stranded DNA-dependent ATPase activities cosedimented in glycerol gradients with two polypeptides of 110 and 90 kDa with a sedimentation coefficient of 7.4 S. The DNA Helicase was markedly stimulated by DNA substrates with a 5'-tailed fork. A DNA substrate with a 3'-tailed fork structure was less stimulatory, although it was more active than substrates without a fork. The directionality of unwinding is 3'-->5' with respect to the single-stranded DNA to which the enzyme was bound. The Helicase activity also required a single-stranded DNA-binding protein (SSB) for unwinding activity. The stimulation by SSBs was nonspecific; all SSBs tested, such as human SSB, bacteriophage T4 gene 32, and Escherichia coli SSB, stimulated the DNA Helicase activity to a varying extent in the presence of a fork structure. With long duplex substrates (> 500 base pairs), the presence of a fork substantially stimulated the DNA Helicase activity in the presence of E. coli SSB. Human SSB stimulated the DNA Helicase activity to the greatest extent (> 10-fold) with a substrate containing a fork compared with substrates without a fork. DNA Helicase activity required ATP hydrolysis and could be supported by all eight nucleoside triphosphates. The Km values for ATP and dATP in unwinding were 28 and 48 microM, respectively. In general, ribonucleoside triphosphates were better effectors than deoxyribonucleoside triphosphates. The properties of this DNA Helicase make it a candidate for a DNA replicative Helicase in human cells.
Paul E. Boehmer - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of a DNA-Helicase by peptide nucleic acids
Nucleic acids research, 1999Co-Authors: Lionel Bastide, Giuseppe Villani, Paul E. Boehmer, Bernard LebleuAbstract:Bis-peptide nucleic acid (bis-PNA) binding results in D-loop formation by strand displacement at complementary homopurine stretches in DNA duplexes. Transcription and replication in intact cells is mediated by multienzymatic complexes involving several proteins other than polymerases. The behaviour of the highly stable clamp structure formed by bis-PNAs has thus far been studied with respect to their capacity to arrest RNA polymerases. Little attention has been given to their recognition and processing by DNA Helicases. In this report we have investigated the inhibitory effect of a bis-PNA on the DNA-Helicase activity of the well characterized herpes simplex type I UL9 protein. Unwinding by UL9 of a synthetic substrate is significantly inhibited by a bis-PNA and the addition of the ICP8 protein, which increases UL9 processivity, does not relieve this inhibition.
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The UL8 Subunit of the Herpes Simplex Virus Type-1 DNA Helicase-Primase Optimizes Utilization of DNA Templates Covered by the Homologous Single-strand DNA-binding Protein ICP8
The Journal of biological chemistry, 1996Co-Authors: Nicolas Gac, Giuseppe Villani, Jean Sébastien Hoffmann, Paul E. BoehmerAbstract:Abstract The herpes simplex virus type-1 DNA Helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA Helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA Helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.
Joan Weliky Conaway - One of the best experts on this subject based on the ideXlab platform.
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A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II
Journal of Biological Chemistry, 1999Co-Authors: Rodney Moreland, Franck Tirode, Qin Yan, Joan Weliky Conaway, Jean-marc Egly, Ronald ConawayAbstract:TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter -proximal sites. TFIIH possesses three known ATP-dependent activities: a 3 3 5 DNA Helicase catalyzed by its XPB subunit, a 5 3 3 DNA Helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) ki-nase activity catalyzed by its CDK7 subunit. In this report , we exploit TFIIH mutants to investigate the contributions of TFIIH DNA Helicase and CTD kinase activities to efficient promoter escape by RNA polymer-ase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA Helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.
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multifunctional rna polymerase ii initiation factor delta from rat liver relationship between carboxyl terminal domain kinase atpase and DNA Helicase activities
Journal of Biological Chemistry, 1993Co-Authors: Hiroaki Serizawa, R C Conaway, Joan Weliky ConawayAbstract:Abstract RNA polymerase II initiation factor delta was previously purified from rat liver and found to possess a closely associated DNA-dependent ATPase activity and a protein kinase activity capable of phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Serizawa, H., Conaway, R.C., and Conaway, J.W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7476-7480). In addition, delta's human homolog, BTF2(TFIIH), was recently shown to have an associated DNA Helicase activity (Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J.H.J., Chambon, P., and Egly, J.-M. (1993) Science 259, 58-63). Here we demonstrate that initiation factor delta also possesses DNA Helicase activity. In addition, we compare the properties of delta's associated CTD kinase, ATPase, and DNA Helicase activities. Whereas the enzymatic properties of ATPase and DNA Helicase are similar and consistent with the possibility that they could function in ATP-dependent activation of the preinitiation complex, ATPase and CTD kinase exhibit significant differences in their nucleotide specificities, responses to DNA effectors, and sensitivities to inhibitors.
Ronald Conaway - One of the best experts on this subject based on the ideXlab platform.
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A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II
Journal of Biological Chemistry, 1999Co-Authors: Rodney Moreland, Franck Tirode, Qin Yan, Joan Weliky Conaway, Jean-marc Egly, Ronald ConawayAbstract:TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter -proximal sites. TFIIH possesses three known ATP-dependent activities: a 3 3 5 DNA Helicase catalyzed by its XPB subunit, a 5 3 3 DNA Helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) ki-nase activity catalyzed by its CDK7 subunit. In this report , we exploit TFIIH mutants to investigate the contributions of TFIIH DNA Helicase and CTD kinase activities to efficient promoter escape by RNA polymer-ase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA Helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.
Narendra Tuteja - One of the best experts on this subject based on the ideXlab platform.
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pea DNA Helicase 45 promotes salinity stress tolerance in ir64 rice with improved yield
Plant Signaling & Behavior, 2012Co-Authors: Ranjan Kumar Sahoo, Sarvajeet Singh Gill, Narendra TutejaAbstract:The Helicases provide duplex unwinding function in an ATP-dependent manner and thereby play important role in almost all the nucleic acids transaction. Since stress reduces the protein synthesis by affecting the cellular gene expression machinery, so it is evident that molecules involved in nucleic acid processing including translation factors/Helicases are likely to be affected. Earlier pea DNA Helicase 45 (PDH45), a homolog of translation initiation factor 4A (eIF4A) was reported to play important role in salinity stress tolerance in tobacco and Bangladeshi rice variety Binnatoa. We report here the overexpression of PDH45 gene in the indica rice variety IR64, via Agrobacterium-mediated transformation. Molecular analysis of the transgenics revealed stable integration of the transgene in the T1 generation. Enhanced tolerance to salinity was observed in the plants transformed with PDH45 gene. Better physiological and yield performances including endogenous nutrient contents (N, P, K, Na) of the transgenics...
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Inhibition of unwinding and ATPase activities of pea MCM6 DNA Helicase by actinomycin and nogalamycin
Plant signaling & behavior, 2011Co-Authors: Ngoc Q. Tran, Renu Tuteja, Xuan Hoi Pham, Narendra TutejaAbstract:Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3′ to 5′ DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities1 (Plant Mol Biol 2010; DOI: 10.1007/s11103-010-9675-7). Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA Helicase. The results show that actinomycin and nogalamycin inhibited the DNA Helicase (apparent Ki values of 10 and 1 µM, respectively) and ATPase (apparent Ki values of 100 and 17 µM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA Helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA Helicase unwinding.
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A single subunit MCM6 from pea forms homohexamer and functions as DNA Helicase
Plant Molecular Biology, 2010Co-Authors: Ngoc Quang Tran, Hung Quang Dang, Renu Tuteja, Narendra TutejaAbstract:The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative Helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3′–5′ DNA Helicase, ATP-binding and ATPase activities. The Helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA Helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA Helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.
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Inhibition of Pea Chloroplast DNA Helicase Unwinding and ATPase Activities by DNA-Interacting Ligands
Biochemical and biophysical research communications, 1998Co-Authors: Narendra Tuteja, Tuan-nghia PhanAbstract:Abstract DNA Helicases unwind the duplex DNA in an ATP dependent manner and thus play an essential role in DNA replication, repair, recombination and transcription. Any DNA-interacting ligand which will modulate DNA Helicase activity may interrupt practically all kinds of DNA transactions. There are no studies on the effect of various cytotoxic DNA-interacting ligands on organelle Helicases. We have determined the effect of camptothecin, VP-16 (etoposide), ellipticine, genistein, novobiocin, m-AMSA, actinomycin C 1 , ethidium bromide, daunorubicin and nogalamycin on unwinding and ATPase activities of purified chloroplast DNA Helicase from pea ( Pisum sativum ). Our study has shown that DNA-intercalating ligands actinomycin C 1 , ethidium bromide, daunorubicin and nogalamycin were inhibiting the DNA unwinding activity with an apparent Ki of 2.9 μM, 3.0 μM, 1.4 μM and 1.0 μM, respectively. These four inhibitors also inhibited the ATPase activity of pea chloroplast DNA Helicase. These results indicate that the intercalation of the inhibitors into DNA generates a complex that impedes the translocation of chloroplast DNA Helicase, resulting in both inhibition of unwinding activity and ATP hydrolysis. This study would be useful for understanding the mechanism of organelle DNA Helicase unwinding and the mechanism by which these DNA-interacting ligands inhibit cellular function.
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Inhibition of DNA unwinding and ATPase activities of human DNA Helicase II by chemotherapeutic agents.
Biochemical and biophysical research communications, 1997Co-Authors: Narendra Tuteja, Renu Tuteja, Tuan-nghia Phan, Alexander Ochem, Arturo FalaschiAbstract:DNA Helicases catalyze the unwinding of duplex DNA and thus play important roles in the processing of DNA, little is known about the effects of various cytotoxic or antitumor chemotherapeutic agents on purified human DNA Helicases. We have determined the effect of actinomycin C1, VP-16, camptothecin, ethidium bromide, ellipticine, nogalamycin, novobiocin, genistein, m-AMSA, aphidicolin and daunorubicin on the enzymatic activities of purified human DNA Helicase II which was identified as Ku autoantigen. Ku contains DNA Helicase, ATPase and DNA end binding activities. Our data have shown that out of several chemotherapeutic agents tested ethidium bromide, actinomycin C1, daunorubicin and nogalamycin were inhibitors of DNA unwinding activity of human DNA Helicase II with ID50 values of 8.44 microM, 11.68 microM, 6.23 microM and 0.42 microM respectively. These inhibitors also inhibited the ATPase activity but not the DNA binding activity of this Helicase. This inhibition could be due to binding of these drugs to DNA, thereby impeding the movement of the Helicase for unwinding action which may be their most important pharmacological function against cancer cells.