Differences in the incorporation of bromodeoxyuridine by human lymphoblastoid cell lines.

Long term human lymphoblastoid lines differ in their ability to grow in medium containing bromodeoxyuridine (BrdU) and to incorporate analog into their DNA. Eight Burkitt's lymphoma cell lines divided at least twice in BrdU-containing medium and made DNA in which over 90% of the thymidine residues were substituted with analog in both strands. Three infectious mononucleosis-derived lines and 24 lines transformed in vitro were inhibited by BrdU after one cell division and made only hybrid DNA in which one strand was substituted with analog. One out of eight normal individuals from whom long term lines were prepared gave cell lines which divided at least twice in BrdU and gave DNA in which both strands were substituted with analog. It would appear that intrinsic cellular factors regulate the response to BrdU and that Burkitt's tumor lines are characterized by their ability to make stable doubly substituted DNA containing a high proportion of halogenated analog.     

Specific DNA cleavage by an analog of netropsin containing a copper(II) chelating peptide Gly-Gly-His]

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.     

Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety

Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied. All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure. The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog. The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity. This agreed with the result of a cross-linking assay. These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.     

Base-pairing models to account for the mutagenicity and specificity of the purine analog 2-amino-N6-hydroxyadenine

Potential base-pairing mechanisms of the purine analog, 2-amino-N6-hydroxyadenine (AHA), with the natural bases of DNA are presented. Base-pairing properties of this analog indicate that AHA may induce transitions and transversions of base pairs in DNA.     

Role of p53 in the ability of 1,2-diaminocyclohexane-diacetato-dichloro-Pt(IV) to circumvent cisplatin resistance

Several reports indicate that the mechanism of resistance to cisplatin is multifactorial. However, DNA damage tolerance appears to be the more significant mechanism. It is clear that resistance in general is a major clinical concern, and a number of approaches have been taken to circumvent this clinical impediment. One approach is through analog development, and we have identified 1,2-diaminocyclohexane-diacetatodichloro-platinum(IV) as an analog with activity in cisplatin resistance. The activity is greatest against ovarian tumor cell lines where the latent, non-inducible wild-type p53 function can be reactivated by the analog. This functional activation of p53 also corresponds to a reduced threshold for tolerance to DNA damage induced by the analog. Interestingly, cell lines with mutant or null p53 are cross-resistant to the analog. The data indicate that cisplatin resistance due to an increase in DNA damage tolerance can arise through a loss of p53 function, and that functional activation of latent wild-type p53 by the analog facilitates cell death and circumvents this resistance mechanism.     

4′-C-Aminomethyl-2′-deoxy-2′-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting the ability of a DNA/RNA duplex to activate RNase H

An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-β-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides.     

Photolytic binding of the monoazido analog of ethidium to yeast mitochondrial DNA: competition by ethidium.

The [14C]-labeled monoazido analog of ethidium, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, when mixed with yeast cells and photolyzed, produced covalent adducts with both nuclear and mitochondrial DNA via the light-generated nitrene. The binding efficiency was about 12 times higher in mitochondrial than nuclear DNA. Moreover, the parent ethidium bromide at a 5-fold excess was an effective competitor for the binding of the monoazide analog with mitochondrial DNA, but not with nuclear DNA.     

Synthesis and property of alpha-oligodeoxynucleotide phosphorothioate.

Unnatural DNA analog, alpha-2'-deoxy-oligoadenylate phosphorothioate bearing 3'-normal monophosphate moiety was conveniently synthesized through phosphoramidite solid support method. The starting material of the DNA analog, alpha-2'-deoxyadenosine, was prepared in a stereospecific manner by the coupling reaction between activated nucleobase and 1-chloro-2-deoxyribose derivative.     

Synthesis and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)

Abstract

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs’ analogs have synthesized by structural changes of nucleoside’s susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl – analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π–π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.     

Effects of 2-chloroadenine substitution in DNA on restriction endonuclease cleavage reactions.

The purine analog, 2-chloro-2'-deoxyadenosine triphosphate (CldATP), was incorporated enzymatically in place of dATP into the minus strand of M13mp18 duplex DNA. Its effect on protein-DNA interactions was assessed by determining the amount of DNA cleavage by type II restriction endonucleases. Substitution of chloroadenine (CIAde) for adenine (Ade) in DNA appreciably decreased the amount and rate of DNA cleavage of the minus strand when the analog was situated within the appropriate endonuclease recognition site. CIAde residues flanking a restriction site had variable effects. SmaI cleaved both CIAde-containing and control substrates with equal efficiency. NarI, however, was stimulated 1.5-fold by the presence of CIAde outside its recognition site. The effects of analog incorporation on restriction enzyme cleavage of an opposing unsubstituted strand of duplex DNA was examined by enzymatically incorporating CIdATP into the complementary minus strand of a 36-base oligonucleotide. Endonucleolytic cleavage of both plus and minus strands was reduced on 36-mers containing CIAde residues located within only the minus strand. These data suggest that CIAde residues incorporated into a single DNA strand may have an appreciable effect on DNA-protein interactions that involve one or both strands of duplex DNA.     

Synthesis and site-specific incorporation of a simple fluorescent pyrimidine

We describe procedures for the synthesis of a fluorescent pyrimidine analog and its site-specific incorporation into a DNA oligomer. The 5'-protected and 3'-activated nucleoside 4 is synthesized in three steps with an overall yield of 40%. Site-specific incorporation into a DNA oligomer occurs with greater than 88% coupling efficiency. This isosteric fluorescent DNA analog can be used to monitor denaturation of DNA duplexes via fluorescence and can positively detect the presence of abasic sites in DNA duplexes. The total time for synthesis of the phosphoramidite 4 is about 75 h, whereas the total time for site-specific incorporation of nucleoside 2 into an oligonucleotide and purification of the corresponding oligonucleotide is about 114 hours.     

A new binary system for photosensitized labeling of DNA polymerases in nuclear extract

A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3"-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5"-phosphate) at the 5"-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase labeling.     

UV-induced cross-linking of Tet repressor to DNA containing tet operator sequences and 8-azidoadenines.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography, proton resonance spectroscopy, infrared spectroscopy and UV spectroscopy. Its photolysis upon UV irradiation was studied. After incorporation of this dATP analog into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor protein by UV photocross-linking becomes possible. Photocross-linking of protein to DNA was demonstrated by the reduced migration of the DNA in SDS polyacrylamide gel electrophoresis. Addition of the inducer tetracycline prior to UV irradiation significantly reduces the DNA-protein cross-linking rate. The long wave UV light applied here does not significantly alter the DNA or the protein under the photocross-linking conditions.     

Synthetic DNA-bindings ligands containing reaction centers specific for AT- and GC-pairs in DNA

In the present communication design, synthesis and DNA binding activities of three bis-netropsins and two netropsin analogs containing two N-propylpyrrolecarboxamide fragments linked covalently to peptides Gly-Gly-(analog I) and Val-Val-Val-Gly-Gly-(analog II) are reported. Each bis-netropsin consists of two netropsin-like fragments attached to peptides -Gly-Cys-Gly-NH2 (compound IIIa), H-Gly-Cys-Gly-Gly-Gly-(compound IV) or Gly-Cys-Sar-NH2 (compound IIIb) which are linked symmetrically via S-S bonds. Physico-chemical studies show that each bis-netropsin carries 6 AT-specific reaction centers and covers approximately 10 base pairs upon binding to poly(dA).poly(dT). This indicates that two netropsin-like fragments of the bis-netropsin molecule are implicated in specific interaction with DNA base pairs. The peptide fragments of bis-netropsins IIIa and IV form small beta-sheets containing two-GC-specific reaction centers. The DNase I cleavage patterns of bis-netropsin-DNA complexes visualized by high resolution gel electrophoresis show that the preferred binding sites for bis-netropsins IIIa and IV are identical and contain two runs of three or more AT pairs separated by two GC pairs. Specificity determinants of netropsin analog II binding in the beta-associated dimeric form are identical to those of bis-netropsin IIIa thereby indicating that there is a similarity in the structure of complexes formed by these ligands with DNA. In the monomeric form analog II exhibits binding specificity identical to that of analog I. Replacement of C-terminal glycine residues by sarcosines in the peptide fragments of bis-netropsin IIIa leads to a decrease in the affinity of ligand for DNA.     

Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.     

Identification of peptides involved in S-adenosylmethionine binding in the EcoRI DNA methylase. Photoaffinity laveling with 8-azido-S-adenosylmethionine

The Mr 38,050 monomeric EcoRI DNA methylase is part of a bacterial restriction-modification system. The methylase transfers the methyl group from S-adenosylmethionine (AdoMet) to the second adenine in the double-stranded DNA sequence 5'-GAATTC-3'. We have used the radiolabeled photoaffinity analog 8-azido-S-adenosylmethionine (8-N3-AdoMet) to identify peptides at the AdoMet binding site in the binary methylase-cofactor analog complex. The dissociation constants in the absence of DNA for the analog and AdoMet are 12.9 and 4.8 microM, respectively. The apparent kcat and Km values, obtained with the double-stranded DNA substrate 5'-CGCGAATTCGCG-3', are 5.0 s-1 and 0.710 microM (8-N3-AdoMet) and 4.3 s-1 and 0.335 microM (AdoMet). Photolabeling by 8-N3-AdoMet occurs upon irradiation with ultraviolet light and is inhibited by AdoMet. Digestion of the adducted methylase with subtilisin generated several radiolabeled peptides. Peptide sequencing from independent photolabeling experiments revealed two radiolabeled peptides containing amino acids 206-212 and 213-221. Instability of the adducted peptides precluded assignment of modified amino acids.     

Quantitative determination of antiviral nucleoside analog in DNA

A technique for the analysis of the amount of an antiviral nucleoside analog incorporated into DNA, utilizing enzymatic digestion of DNA, followed by high-performance liquid chromatography is described. The cells or tissue samples were treated with perchloric acid to inactivate the nucleases, then digested with pronase in the presence of EDTA. DNA was purified by CsCl centrifugation followed by Sephadex chromatography and treatment with deoxyribonuclease 1 and venom phosphodiesterase. The deoxyribonucleoside monophosphates and the monophosphate of the nucleoside analog liberated from DNA were separated and quantitated by HPLC analysis and measurement of radioactivity. This assay is more sensitive, specific, and precise than the determination of DNA density shift. It is also applicable for nucleoside analogs which do not change the density of DNA either because of their structure or their very small degree of incorporation.     

Triostin A derived hybrid for simultaneous DNA binding and metal coordination

The natural product triostin A is known as an antibiotic based on specific DNA recognition. Structurally, a bicyclic depsipeptide backbone provides a well-defined scaffold preorganizing the recognition motifs for bisintercalation. Replacing the intercalating quinoxaline moieties of triostin A by nucleobases results in a potential major groove binder. The functionalization of this DNA binding triostin A analog with a metal binding ligand system is reported, thereby generating a hybrid molecule with DNA binding and metal coordinating capability. Transition metal ions can be placed in close proximity to dsDNA by means of non-covalent interactions. The synthesis of the nucleobase-modified triostin A analog is described containing a propargylglycine for later attachment of the ligand by click-chemistry. As ligand, two [1,4,7]triazacyclononane rings were bridged by a phenol. Formation of the proposed binuclear zinc complex was confirmed for the ligand and the triostin A analog/ligand construct by high-resolution mass spectrometry. The complex as well as the respective hybrid led to stabilization of dsDNA, thus implying that metal complexation and DNA binding are independent processes.     

The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases.

The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.     

Incorporation of 5-bromo-2′deoxyuridine (BUdR) in Dictyostelium discoideum DNA

The modality of incorporation of 5-bromo-2′deoxyuridine (BUdR) in the DNA of Dictyostelium discoideum was studied after one generation of growth of the amoebae in the presence of different concentrations of the drug. The analog was incorporated following the semiconservative pattern of DNA replication. BUdR incorporation in monosubstituted DNA has been measured both by CsCl isopycnic centrifugation or by base analysis chromatography; substitution of thymidine by its analog reaches a maximal value of 30% (60% in the substituted strand). Up to 20% substitution it is proportional to the drug concentration in the growth medium. In these conditions, thymidine substitution is higher in repetitive sequences of the DNA than in unique sequences; the percent of increase of thymidine substitution in repetitive fractions versus total DNA is inversely proportional to thymidine substitution in total DNA.