Base incorporation and extension at a site-specific ethenocytosine by Escherichia coli DNA polymerase I Klenow fragment

Simha, Devendranath; Yadav, Deepmala; Rzepka, Robert W.; Palejwala, Vaseem A.; Humayun, M. Zafri

doi:10.1016/0027-5107(94)90219-4

Cited by 6 publications

(5 citation statements)

References 12 publications

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“…Inhibition of DNA Synthesis by the ⑀C⅐ANPG ComplexWhen present in DNA, ⑀C can be bypassed by DNA polymerases (46,47); however, we speculated that ANPG bound to ⑀C could block DNA synthesis. As shown in Fig.…”

Section: Fig 2 Analysis Of Anpg⅐⑀c Interactionmentioning

confidence: 94%

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

Gros¹,

Maksimenko

Privezentzev³

et al. 2004

Journal of Biological Chemistry

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Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(⑀)-adducts. E-bases have been implicated in vinyl chlorideinduced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N 4 -ethenocytosine (⑀C) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to ⑀C when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the ⑀C⅐ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the ⑀C⅐ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that ⑀C could be more genotoxic than 1,N 6 -ethenoadenine (⑀A) residues in vivo. The proposed model of ANPG-mediated genotoxicity of ⑀C provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.

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Section: Fig 2 Analysis Of Anpg⅐⑀c Interactionmentioning

confidence: 94%

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

Gros¹,

Maksimenko

Privezentzev³

et al. 2004

Journal of Biological Chemistry

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“…Exocyclic base modifications, such as C, can lead to genetic mutations due to possible alteration of coding information at the lesion site. In this regard, several polymerases show a tendency to add an A or T residue to the nascent strand across from an C site (9)(10)(11)(12)(13)(14)(15), thereby resulting in either a C to T transition or a C to A transversion. Addition of G and C across from C, and even U with RNA polymerase, as well as one-and two-base deletions also have been observed, with the specific outcome depending on the polymerase studied (13,15,16).…”

mentioning

confidence: 99%

“…In this regard, several polymerases show a tendency to add an A or T residue to the nascent strand across from an C site (9)(10)(11)(12)(13)(14)(15), thereby resulting in either a C to T transition or a C to A transversion. Addition of G and C across from C, and even U with RNA polymerase, as well as one-and two-base deletions also have been observed, with the specific outcome depending on the polymerase studied (13,15,16). In vivo mutation rates resulting from C depend strongly on the organism, with a mutation frequency range of approximately 2% in bacteria (11,12), to over 80% in mammalian COS cells (12).…”

mentioning

confidence: 99%

The Impact of an Exocyclic Cytosine Adduct on DNA Duplex Properties: Significant Thermodynamic Consequences Despite Modest Lesion-Induced Structural Alterations

et al. 1998

Biochemistry

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The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.

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“…1993b) as well as by certain endogenous mutagens (CHEN and CHUNC 1994). As predicted by the etheno bridging over two base pairing positions (Figure l ) , EC has been shown to have the in vitro (SIMHA et al 1991(SIMHA et al , 1994 and in vivo (JACOBSEN et al 1989;JACOBSEN and HUMAWN 1990;PALEJWALA et al 1991PALEJWALA et al , 1993bPALEJWALA et al , 1994BASU et al 1993;MORWA et al 1994) template characteristics of a noninstructive lesion. In the experiments described here, the…”

Section: Resultsmentioning

confidence: 85%

“…In vitro primer elongation experiments by E. coli DNA polymerase I large Klenow fragment on an oligonucleotide template bearing a site-specific EC residue does not reveal an ability to code for guanine. Instead, in this system, EC residues display the template characteristics a classical noninstructive lesions in that there is inefficient base misinsertion that appears to follow the adenine rule (SIMHA et al 1991(SIMHA et al ,1994. (Center) Strategy used for incorporating a sitespecific EC residue into M13 mp7L2 DNA.…”

Section: -Mermentioning

confidence: 99%

Alkylating agents induce UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli.

Wang¹,

Palejwala²,

Dunman³

et al. 1995

Genetics

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Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing delta recA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.

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Base incorporation and extension at a site-specific ethenocytosine by Escherichia coli DNA polymerase I Klenow fragment

Cited by 6 publications

References 12 publications

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

The Impact of an Exocyclic Cytosine Adduct on DNA Duplex Properties: Significant Thermodynamic Consequences Despite Modest Lesion-Induced Structural Alterations

Alkylating agents induce UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli.

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