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O S Bhanot

Publications and source records attributed to O S Bhanot.

17 recordsLinked to original sources
PubMed1994

The role of 3-hydroxyethyldeoxyuridine in mutagenesis by ethylene oxide.

Ethylene oxide, a direct-acting mutagen and carcinogen, produces 3-hydroxyethyldeoxyuridine (3-HE-dU) after initial alkylation at N3 of dC, followed by rapid hydrolytic deamination. The significance of formation of 3-HE-dU in DNA was investigated by in vitro DNA replication of 3-HE-dU. A 55-nucleotide DNA template, containing 3-HE-dU at a single site, was constructed. DNA products, synthesized on the site-modified template, were analyzed and mutagenic bypass at 3-HE-dU estimated. The 3-HE-dU lesion blocked DNA replication by the Klenow fragment of Escherichia coli polymerase I (Kf Pol I) and bacteriophage T7 polymerase (T7 Pol) 3' to 3-HE-dU and after incorporating a nucleotide opposite 3-HE-dU. DNA synthesis past 3-HE-dU was negligible (< 3%). Substitution of Kf Pol I (exo-) and T7 Pol (exo-), polymerases lacking 3'-->5' exonuclease proofreading activity, for Kf Pol I and T7 Pol, respectively, facilitated DNA synthesis past 3-HE-dU. The bypass synthesis by Kf Pol I (exo-) was 60% and 90% by T7 Pol (exo-). These results suggest that the 3-HE-dU lesion could be bypassed, but that the extension at 3-HE-dU is rate-limiting. In the absence of proofreading, the nucleotide incorporated opposite 3-HE-dU is not excised and remains in position long enough for extension to occur. During post-lesion synthesis, both dA and dT were incorporated opposite 3-HE-dU. Since 3-HE-dU is derived from dC alkylation by ethylene oxide, incorporation of dA and dT opposite 3-HE-dU implicates this lesion in G.C-->A.T and G.C-->T.A mutagenesis.

Base Sequence↗
PubMed1994

UVM, an ultraviolet-inducible RecA-independent mutagenic phenomenon in Escherichia coli.

Most mutagenic DNA lesions are noninstructive in the sense that template instruction is either missing or inaccessible during DNA replication, leading to replication arrest. According to the SOS hypothesis, arrested replication induces the expression of SOS factors that force replication past stalled sites at the cost of mutagenesis. We have recently shown that prior UV irradiation of delta recA cells, in which the SOS pathway does not function, enhances mutagenesis at an ethenocytosine residue borne on a circular gapped duplex DNA vector, indicating the existence of an SOS-independent inducible mutagenic phenomenon termed UVM (UV modulation of mutagenesis). In the previous experiments, mutation fixation was expected to occur during gap-filling DNA synthesis. To test whether UVM is observable during normal replication by DNA polymerase III, we have examined mutagenesis at an epsilon C residue borne on M13 single-stranded DNA. By analyzing mutation frequency and specificity using a multiplex sequence assay, we now show that UVM is observable in UV-irradiated recA+, and in delta recA cells. These data indicate that UV irradiation induces a previously unrecognized mutagenic mechanism in Escherichia coli, and that this mechanism is manifested during gap-filling DNA synthesis as well as during normal DNA replication.

Base Sequence↗
PubMed1994

The role of mutagenic metal ions in mediating in vitro mispairing by alkylpyrimidines.

A variety of alkylating mutagens and carcinogens produce pyrimidine adducts in DNA that block DNA synthesis in vitro. Since DNA synthesis past the lesion is a necessary step to produce mutations, we investigated the role of the mutagenic metal ion Mn++ in facilitating DNA synthesis past alkylpyrimidines. In the presence of the natural metal activator Mg++, N3-ethyldeoxythymidine (N3-Et-dT) and O2-ethyldeoxythymidine (O2-Et-dT), present at a single site in DNA, blocked in vitro DNA synthesis 3' to the lesion and after incorporating dA opposite each lesion. The presence of Mn++ permitted postlesion synthesis with dT misincorporated opposite N3-Et-dT and O2-Et-dT, implicating these lesions in A.T-->T.A transversion mutagenesis. The DNA synthesis block by O4-ethyldeoxythymidine (O4-Et-dT) in the presence of Mg++ was partial and was also removed by Mn++. Consistent with in vivo studies, dG was incorporated opposite O4-Et-dT during postlesion synthesis, leading to A.T-->G.C transition mutagenesis. We also have discovered a new class of DNA adducts, N3-hydroxyalkyldeoxyuridine (3-HA-dU) lesions, which are produced by mutagenic and carcinogenic aliphatic epoxides. 3-HA-dU is formed after initial alkylation at the N3 position of dC followed by a rapid hydrolytic deamination. As observed with the analogous mutagenic N3-Et-dT, the ethylene oxide-induced 3-hydroxyethyldeoxyuridine (3-HE-dU) blocked in vitro DNA synthesis, which could be by-passed in the presence of Mn++. The nucleotide incorporated opposite 3-HE-dU during postlesion synthesis is being identified. These studies suggest a role for Mn++ in mediating mutagenic and carcinogenic effects of environmentally important ethylating agents and aliphatic epoxides.

Alkylation↗
PubMed1992

In vitro mispairing specificity of O2-ethylthymidine.

The O2-position of thymine is a major site of base alkylation by N-nitroso-alkylating agents, and its biological relevance remains obscure. The potential significance of this DNA damage was ascertained by studying in vitro DNA replication properties of O2-ethylthymidine (O2-Et-dT) site-specifically incorporated into a 36-nucleotide template. DNA replication was initiated eight nucleotides away from the O2-Et-dT lesion by Escherichia coli polymerase I (Klenow fragment) using a 17-nucleotide primer. In the presence of 10 microM dNTP and Mg2+, O2-Et-dT blocked DNA replication predominantly (94%) 3' to O2-Et-dT, with the remainder (5%) blocked after incorporation of a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). Postlesion synthesis was negligible (less than 1%). Nucleotide incorporation opposite O2-Et-dT increased to 23% at 200 microM dNTP. Postlesion synthesis remained negligible (less than 2%). DNA sequencing revealed dA present opposite O2-Et-dT in the incorporation-dependent blocked product. Negligible postlesion synthesis suggests that incorporation of dA opposite O2-Et-dT inhibits in vitro DNA synthesis. The O2-Et-dT.dA base pair may also impede DNA synthesis in vivo, contributing to the cytotoxicity of the ethylating agents. Substitution of Mn2+ for Mg2+ enhanced nucleotide incorporation opposite O2-Et-dT and produced postlesion synthesis (16%) at 10 microM dNTP, which increased to 39% at 200 microM dNTP. DNA sequence analysis showed that while dA was present opposite O2-Et-dT in the incorporation-dependent blocked product, both dA and dT were present opposite this lesion in the postlesion synthesis product.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents.

A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.

Base Sequence↗
PubMed1991

The role of N3-ethyldeoxythymidine in mutagenesis and cytotoxicity by ethylating agents.

The significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine was investigated using an in vitro DNA replication system. The system utilized a primed template in which the 3'-end of the primer is eight nucleotides away from N3-ethyldeoxythymidine (N3-Et-dT), present at template position 26 from the 3'-end. The 34-nucleotide template corresponds to a specific DNA sequence at gene G of bacteriophage phi X174. DNA synthesis products were quantitated by electrophoretic separation and autoradiography. At 10 microM dNTP and 0.5 mM Mn2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 60% after incorporating a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product) and 39% 3' to N3-Et-dT. DNA replication past the lesion (post-lesion synthesis) was negligible. Post-lesion synthesis increased using higher concentrations of dNTP, reaching 68% at 200 microM dNTP. DNA sequencing revealed that dA was incorporated opposite N3-Et-dT in the incorporation-dependent blocked product. In the post-lesion synthesis product, dT was exclusively incorporated opposite N3-Et-dT. Formation of the N3-Et-dT.dA base pair at the replication fork terminated DNA synthesis, while the N3-Et-dT.dT base pair formed at the 3'-end of the growing chain was extended, leading to an A.T----T.A transversion mutation. The results suggest a dual role for the N3-Et-dT lesion, contributing in part to the cytotoxicity and mutagenicity of ethylating agents. These studies provide a basis for understanding the activation of oncogene neu by A.T----T.A transversion mutation in rat neuroblastomas induced by N-ethyl-N-nitrosourea.

Alkylating Agents↗
PubMed1990

Incorporation of dA opposite N3-ethylthymidine terminates in vitro DNA synthesis.

N3-Ethylthymidine (N3-Et-dT) was site specifically incorporated into a 17-nucleotide oligomer to investigate the significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine. The 5'-(dimethoxytrityl)-protected N3-Et-dT was converted to the corresponding 3'-phosphoramidite and used to incorporate N3-Et-dT at a single site in the oligonucleotide during synthesis by the phosphite triester method. The purified N3-Et-dT-containing oligomer was ligated to a second 17-mer to yield a 34-nucleotide template with N3-Et-dT present at position 26 from the 3'-end. The template DNA, which corresponds to a specific sequence at gene G of bacteriophage phi X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT. At 10 microM dNTP and 5 mM Mg2+, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96% immediately 3' to N3-Et-dT and 4% after incorporation of a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product). DNA replication past the lesion (postlesion synthesis) was negligible. Incorporation opposite N3-Et-dT increased with increased dNTP concentrations, reaching 35% at 200 microM. Postlesion synthesis remained negligible. DNA sequencing of the incorporation-dependent blocked product revealed that dA is incorporated opposite N3-Et-dT consistent with the "A" rule in mutagenesis. Formation of the N3-Et-dT.dA base pair at the 3'-end of the growing chain terminated DNA synthesis. These results implicate N3-Et-dT as a potentially cytotoxic lesion produced by ethylating agents.

Bacteriophage phi X 174↗
PubMed1986

The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.

A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.

Bacteriophage phi X 174↗
PubMed1982

The effect of bisulfite-induced C to U transitions on aminoacylation of Escherichia coli glycine tRNA.

The effect of bisulfite-induced C to U transitions on the aminoacylation of highly purified Escherichia coli tRNAgly3,gcc has been studied. On treatment with 3.2 M NaHSO3 at 25 degrees C and pH 5.8, C to U transitions occurred at 4 of the 21 cytosine residues during the first 16 h. After about 12 h, 2 additional unidentified residues begin to react. From these data and theoretical consideration, we conclude: 1) E. coli tRNAgly3,gcc exists in the native conformation under the conditions of the reaction; 2) its ordered structure is similar to yeast tRNAPhe; 3) its anticodon loop is flexible in solution. During the reaction, loss of glycine acceptor activity followed first order kinetics with a t1/2 = 1.8 h. After modification for 1.25 h and aminoacylation (61% glycine acceptor activity remaining), the aminoacylated fraction was isolated. The fractional change at each of the reactive residues in the unfractionated mixture (f) and the aminoacylated fraction (p) was measured. The results were: C35, f = 0.33, p = 0.19; C36, f = 0.25, p = 0.10; C74, f = 0.28, p = 0.26; C75, f = 0.25, p = 0.20. From these data, the Modulation Constant for each reactive residue was calculated from the equation M = (f - p)/f(1 - p): M35 (anticodon) = 0.52; M36 (anticodon) = 0.67; M74 (CCA end) = 0.10; M75 (CCA end) = 0.25. These values, which are based on the assumptions that the C to U reactions occur independently and that the effect of each change on the acceptor activity is an independent event, express the fractional loss in activity that would occur from a C to U change at the residue in question by itself. From these results, we conclude: 1) approximately 80% of the observed inactivation was due to changes in the anticodon; 2) neither of these anticodon residues (C35 and C36) is essential for aminoacylation; 3) a C to U change at C75 (CCA end) has a small effect on aminoacylation; 4) a C to U change at C74 (CCA end) has little or no effect on aminoacylation.

Cytosine↗
PubMed1979

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.

Bacteriophage phi X 174↗
PubMed1977

Isolation and characterization of valine transfer RNA from Saccharomyces cerevisiae.

Two procedures for isolating valine tRNA from commercial bakers' yeast were investigated. The first involved: (a) counter double current distribution; (b) chromatography on benzoyl-DEAE-cellulose; (c) reverse phase chromatography on Chromosorb G saturated with trioctylpropylammonium bromide (Oakridge System 3). The material isolated lacked the 3'-terminal adenylic acid residue. The second procedure involved the first two steps above followed by: (a) enzymatic aminoacylation with a partially purified yeast extract; (b) derivatization with N-phenoxyacetoxysuccinimide; (c) chromatography on benzoyl-DEAE-cellulose; (d) reverse phase chromatography, System 3. The product was intact tRNA. It was a mixture of isoacceptors (59:41) differing by a modification (uracil leads to dihydrouracil) at position 48. It was free of denatured material; specific activity 1,825 pmol of valine/A260 unit of tRNA. Sequence analysis confirmed the recently corrected structure (Bonnet, J., Ebel, J. P., Dirheimer, G., Shershneva, L. P., Krutilina, A. I., Venkstern, T. V., and Bayev, A. A. (1974) Biochimie 56, 1211-1213). A preliminary study of the alkaline hydrolysis of the 7-methylguanosine residue that occurs at position 47 showed that at least two products are formed instead of only one as usually quoted in the literature. A rapid, ultramicro, chromatographic system for separating these products and measuring them quantitatively is described.

Guanosine↗
PubMed1977

Bisulfite-induced C changed to U transitions in yeast alanine tRNA.

The reaction of yeast tRNAAla1ab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Five reactive residues have been located. Four of these (C-17 in Loop I, C-36 in the anticodon, C-74 and C-75 near the acceptor end) react to the same extent (42%) under the conditions of the experiment. The other (C-72 in the first base pair of the acceptor stem) reacts much more slowly (8%). No other changes were detected, but kinetic data suggest two or more additional residues may react very slowly. The C changed to U change in the anticodon (igc changed to igu) is a missense change (Ala changed to Thr). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. The quantitative changes for tRNAAla indicate that the stacking lifetimes of C-17, C-36, C-74, and C-75 are about equal. All other cytidine residues are much more tightly stacked. These results are consistent with the folded cloverleaf models that have been proposed from x-ray diffraction studies of yeast tRNAPhe. Residues 48 and 56, which are in single-stranded regions in the unfolded cloverleaf structure, do not react suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also indicate that the anticodon loop is flexible in solution.

Alanine↗
PubMed1977

Bisulfite-induced C changed to U transitions in yeast valine tRNA.

The reaction of yeast tRNAVallab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Six reactive residues have been located. C-17 in loop I is the most reactive (51% conversion) and C-73 in the first base pair of the acceptor stem the least reactive (8%). Three of the remaining reactive residues (C-39 in loop II, C-75 and C-76 near the acceptor stem) react to the same extent (36 to 38%) under the conditions of the experiment. C-37 in the anticodon reacted to a lesser extent (28%) than C-39 (36%), located just 2 residues away in the anticodon loop. No other changes were detected, but kinetic data suggest one or more additional residues may react very slowly. The C changed to U change in the anticodon (iac changed to iau) is a missense change (Val changed to Ile). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. We interpret the quantitative changes in tRNAVal to indicate that C-17 spends a large portion of its lifetime in an unstacked conformation. The stacking lifetimes of C-37, C-39, C-75, and C-76 seem to be similar but not identical. All other cytidine residues are much more tightly stacked. These results are consistent with the folded cloverleaf models that have been proposed from x-ray diffraction studies of yeast tRNAPhe. Residues C-46, C-49, C-57, and C-61, which are present in the single-stranded regions of the unfolded cloverleaf structure, do not react, suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also suggest that anticodon-loop conformations other than the extremes with five bases stacked on either the 3' or 5' portion of the anticodon stem exist in solution and that the anticodon loop is flexible.

Base Sequence↗