共查询到20条相似文献,搜索用时 15 毫秒
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Alessia Stornetta Dr. Todor Angelov Prof. Dr. F. Peter Guengerich Prof. Dr. Shana J. Sturla 《Chembiochem : a European journal of chemical biology》2013,14(13):1634-1639
O6‐Methylguanine (O6‐MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low‐fidelity Y‐family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y‐family polymerases bypass DNA adducts. Previous work showed that Dpo4‐mediated dTTP incorporation is favored opposite O6‐MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O6‐MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O6‐MeG in a DNA template. To this end, we utilized a new fluorescence‐based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O6‐MeG by Dpo4. In single‐dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O6‐MeG. Moreover, pyrimidine‐based dNTPs were generally better incorporated than purine‐based syn‐conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2‐position of dCTP increase the selectivity for incorporation opposite O6‐MeG without a significant loss of efficiency. 相似文献
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Dr. Francis P. McManus Prof. Christopher J. Wilds 《Chembiochem : a European journal of chemical biology》2014,15(13):1966-1977
O6‐Alkylguanine‐DNA alkyltransferases (AGTs) are responsible for the removal of O6‐alkyl 2′‐deoxyguanosine (dG) and O4‐alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O6‐alkylguanine‐DNA‐alkyltransferase) protein, which can repair a range of O4‐alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O4‐alkyl 2′‐deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada‐C and OGT), human AGT, and an OGT/hAGT chimera to remove O4‐methyl and larger adducts (4‐hydroxybutyl and 7‐hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O4‐methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O4 atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct‐containing oligonucleotides and the amounts of repair measured. 相似文献
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O4‐Alkylated‐2‐Deoxyuridine Repair by O6‐Alkylguanine DNA Alkyltransferase is Augmented by a C5‐Fluorine Modification 下载免费PDF全文
Lauralicia Sacre Dr. Derek K. O'Flaherty Philippe Archambault William Copp Prof. Dr. Gilles H. Peslherbe Prof. Dr. Heidi M. Muchall Prof. Dr. Christopher J. Wilds 《Chembiochem : a European journal of chemical biology》2018,19(6):575-582
Oligonucleotides containing various adducts, including ethyl, benzyl, 4‐hydroxybutyl and 7‐hydroxyheptyl groups, at the O4 atom of 5‐fluoro‐O4‐alkyl‐2′‐deoxyuridine were prepared by solid‐phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5‐fluoro‐2′‐deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B‐form DNA structure. O6‐Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5‐fluoro‐O4‐benzyl‐2′‐deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O4‐ethyl and benzyl adducts of 5‐fluoro‐2‐deoxyuridine. Computational assessment of N1‐methyl analogues of the O4‐alkylated nucleobases revealed that the C5‐fluorine modification had an influence on reducing the electron density of the O4?Cα bond, relative to thymine (C5‐methyl) and uracil (C5‐hydrogen). These results reveal the positive influence of the C5‐fluorine atom on the repair of larger O4‐alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT. 相似文献
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Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7‐Substituted 7‐Deazaguanine Nucleobases 下载免费PDF全文
Michaela Mačková Soňa Boháčová Dr. Pavla Perlíková Dr. Lenka Poštová Slavětínská Prof. Dr. Michal Hocek 《Chembiochem : a European journal of chemical biology》2015,16(15):2225-2236
Previous studies of polymerase synthesis of base‐modified DNAs and their cleavage by restriction enzymes have mostly related only to 5‐substituted pyrimidine and 7‐substituted 7‐deazaadenine nucleotides. Here we report the synthesis of a series of 7‐substituted 7‐deazaguanine 2′‐deoxyribonucleoside 5′‐O‐triphosphates (dGRTPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dGRTPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7‐substituted 7‐deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage. 相似文献
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Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases 下载免费PDF全文
Dr. Katharina Höfler Ivo Sarac Prof. Dr. Chris Meier 《Chembiochem : a European journal of chemical biology》2015,16(14):2046-2053
C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis. 相似文献
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Mechanism of Error‐Free Bypass of the Environmental Carcinogen N‐(2′‐Deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone Adduct by Human DNA Polymerase η 下载免费PDF全文
Dr. Amritraj Patra Dr. Dustin A. Politica Arindom Chatterjee E. John Tokarsky Prof. Zucai Suo Prof. Ashis K. Basu Prof. Michael P. Stone Prof. Martin Egli 《Chembiochem : a European journal of chemical biology》2016,17(21):2033-2037
The environmental pollutant 3‐nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8‐dG‐ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error‐free manner by the human Y‐family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol?DNA?dCTP complex between a C8‐dG‐ABA‐containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error‐free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra‐nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson–Crick pair with the incoming dCTP. 相似文献
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Elizabeth Brunk Dr. Birgit Mollwitz Prof. Dr. Ursula Rothlisberger 《Chembiochem : a European journal of chemical biology》2013,14(6):703-710
O6‐alkylguanine‐DNA alkyltransferase (AGT) adopts a non‐enzymatic suicide mechanism for the repair of methylated guanine bases by transferring the methyl adduct to itself, thereby initiating unfolding and fast degradation. Classical molecular dynamics simulations provide quantitative evidence that two conserved glycine residues at the centre of an α‐helix make the structure susceptible to structural perturbations. The stability of this helix, designated the “recognition helix”, is an important factor during the early onset of unfolding of human AGT (hAGT). By combining theory and experiment, we found that helical stability is controlled by key factors in the surrounding protein structure. By using a “double‐clip” mechanism, nearby residues hydrogen bond to both the base and centre of the helix. This double clip stabilises this site in the protein in the absence of substrate, but the helix is destabilised upon alkylation. The present investigation aimed to establish why alkylation of hAGT leads to conformational changes and how the protein environment functions as a switch, thus turning the stability of the protein “on” or “off” to tune degradability. 相似文献
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Viruthachalam Thiagarajan Arivazhagan Rajendran Hiroyuki Satake Seiichi Nishizawa Norio Teramae 《Chembiochem : a European journal of chemical biology》2010,11(1):94-100
The binding behavior of green fluorescent ligands, derivatives of 7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N1‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)propane‐1,3‐diamine (NBD‐NH2) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52×106 M ?1. From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD‐NH2 is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2‐amino‐6,7‐dimethyl‐4‐hydroxypteridine (diMe‐pteridine). 相似文献
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Structural Insights into the Recognition of N2‐Aryl‐ and C8‐Aryl DNA Lesions by the Repair Protein XPA/Rad14 下载免费PDF全文
Charlotte Ebert Nina Simon Dr. Sabine Schneider Prof. Dr. Thomas Carell 《Chembiochem : a European journal of chemical biology》2017,18(14):1379-1382
Aromatic amines are strongly carcinogenic. They are activated in the liver to give reactive nitrenium ions that react with nucleobases within the DNA duplex. The reaction occurs predominantly at the C8 position of the dG base, thereby giving C8‐acetyl‐aryl‐ or C8‐aryl‐dG adducts in an electrophilic aromatic substitution reaction. Alternatively, reaction with the exocyclic 2‐NH2 group is observed. Although the C8 adducts retain base‐pairing properties, base pairing is strongly compromised in the case of the N2 adducts. Here we show crystal structures of two DNA lesions, N2‐acetylnaphthyl‐dG and C8‐fluorenyl‐dG, within a DNA duplex recognized by the repair protein Rad14. The structures confirm that two molecules of the repair protein recognize the lesion and induce a 72 or 78° kink at the site of the damage. Importantly, the same overall kinked structure is induced by binding of the repair proteins, although the structurally different lesions result in distinct stacking interactions of the lesions within the duplex. The results suggest that the repair protein XPA/Rad14 is a sensor that recognizes flexibility. The protein converts the information that structurally different lesions are present in the duplex into a unifying sharply kinked recognition motif. 相似文献
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Sequence‐Specific DNA Photosplitting of Crosslinked DNAs Containing the 3‐Cyanovinylcarbazole Nucleoside by Using DNA Strand Displacement 下载免费PDF全文
Dr. Shigetaka Nakamura Hayato Kawabata Prof. Dr. Kenzo Fujimoto 《Chembiochem : a European journal of chemical biology》2016,17(16):1499-1503
An oligodeoxynucleotide (ODN) containing the ultrafast reversible 3‐cyanovinylcarbazole (CNVK) photo‐crosslinker was photo‐crosslinked to a complementary strand upon exposure to 366 nm irradiation and photosplit by use of 312 nm irradiation. In this paper we report that the photoreaction of CNVK on irradiation at 366 nm involves a photostationary state and that its reaction can be controlled by temperature. Guided by this new insight, we proposed and have now demonstrated previously unknown photosplitting of CNVK aided by DNA strand displacement as an alternative to heating. The photo‐crosslinked double‐stranded DNA (dsDNA) underwent >80 % photosplitting aided by DNA strand displacement on irradiation at 366 nm without heating. In this photosplitting based on DNA strand displacement, the relative thermal stability of the invader strand with respect to the template strands plays an important role, and an invader strand/template strand system that is more stable than the passenger strand/template strand system induces photosplitting without heating. This new strand‐displacement‐aided photosplitting occurred in a sequence‐specific manner through irradiation at 366 nm in the presence of an invader strand. 相似文献
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Mechanism‐Based Inhibitor of DNA Cytosine‐5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2‐Amino‐4‐Halopyridine‐C‐Nucleoside 下载免费PDF全文
Dr. Kousuke Sato Yuma Kunitomo Yukiko Kasai Shohei Utsumi Prof. Isao Suetake Prof. Shoji Tajima Prof. Satoshi Ichikawa Prof. Akira Matsuda 《Chembiochem : a European journal of chemical biology》2018,19(8):865-872
In chromatin, 5‐methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine‐5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐4‐halopyridine‐C‐nucleosides (dXP) and oligodeoxyribonucleotides (ODNs) containing dXP as a novel mechanism‐based inhibitor of DNMTs. The designed ODN containing XPpG forms a complex with DNMTs by covalent bonding through a nucleophilic aromatic substitution (SNAr) reaction, and its cell proliferation activity is investigated. This study suggests that dXP in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chemical probe for studies of epigenetics. Our molecular design using a SNAr reaction would be useful for DNMTs and other protein–DNA interactions. 相似文献
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Molecular Design of an Environmentally Sensitive Fluorescent Nucleoside, 3‐Deaza‐2′‐Deoxyadenosine Derivative: Distinguishing Thymine by Probing the DNA Minor Groove 下载免费PDF全文
Azusa Suzuki Takumi Yanaba Prof. Isao Saito Prof. Yoshio Saito 《Chembiochem : a European journal of chemical biology》2014,15(11):1638-1644
An environmentally sensitive fluorescent nucleoside containing a 3‐deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3‐naphthylethynylated 3‐deaza‐2′‐deoxyadenosine (3nzA, 1 ) exhibited dual fluorescence emission from an intramolecular charge‐transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength. 相似文献
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Hideshi Yokoyama Ryuta Mizutani 《International journal of molecular sciences》2014,15(11):20321-20338
Exposure to the ultraviolet component of sunlight causes DNA damage, which subsequently leads to mutations, cellular transformation, and cell death. DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts are more mutagenic than cyclobutane pyrimidine dimers. These lesions must be repaired because of the high mutagenic potential of (6-4) photoproducts. We here reviewed the structures of (6-4) photoproducts, particularly the detailed structures of the (6-4) lesion and (6-4) lesion-containing double-stranded DNA. We also focused on interactions with their binding proteins such as antibody Fabs, (6-4) photolyase, and nucleotide excision repair protein. The (6-4) photoproducts that bound to these proteins had common structural features: The 5''-side thymine and 3''-side pyrimidone bases of the T(6-4)T segment were in half-chair and planar conformations, respectively, and both bases were positioned nearly perpendicularly to each other. Interactions with binding proteins showed that the DNA helices flanking the T(6-4)T segment were largely kinked, and the flipped-out T(6-4)T segment was recognized by these proteins. These proteins had distinctive binding-site structures that were appropriate for their functions. 相似文献