Heat-induced depyrimidination of deoxyribonucleic acid in neutral solution

Lindahl, Tomas; Karlström, Olle

doi:10.1021/bi00749a020

Cited by 205 publications

(130 citation statements)

References 20 publications

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“…A 20-l reaction used for polymorphism analysis of the MTHFR gene in this work contains approximately 5 ϫ 10 12 primary probes, of which only 2 ϫ 10 6 molecules, or less then one millionth, are cleaved in a 4-h reaction with 600 target molecules (when ␣ 1 ϭ 15 min Ϫ1 ). If under these conditions the rates of formation of abasic sites and the subsequent strand breakage at these abasic sites in a DNA molecule are Ϸ10 Ϫ9 sec Ϫ1 (16,17) and Ϸ5 ϫ 10 Ϫ5 sec Ϫ1 (18), respectively, thermal instability would generate Ϸ2 ϫ 10 7 5Ј arm molecules (19,20), which is an order of magnitude higher than the number of specifically cleaved primary probes. If amplified in the secondary reaction, these breakdown products would make target detection at the level of 600 molecules almost impossible.…”

Section: Discussionmentioning

confidence: 99%

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Hall

Eis

Law

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

231

151

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The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10 7 reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

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Section: Discussionmentioning

confidence: 99%

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Hall

Eis

Law

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

231

151

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“…We have previously analyzed apurinic/apyrimidinic endonuclease activity in extracts from normal cells and from XP cells (7). This enzyme is thought to be involved in the 123 excision of apurinic or apyrimidinic sites from the DNA, some of which are probably generated by the removal of uracil from DNA by uracil DNA N-glycosidase (22,23). Apurinic/apyrimidinic endonuclease is present at a similar high level of activity as uracil DNA N-glycosidase, but as noted above it has an apparent Km for substrate that is more than 100-fold lower.…”

Section: Discussionmentioning

confidence: 99%

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

Kühnlein¹,

Lee²,

Linn³

1978

Nucl Acids Res

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Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblast lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of compl ementati on groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal. INTRODUCTIONUrazil residues apparently can be introduced into DNA by two mechanisms: the spontaneous deamination of DNA-cytosine, or the incorporation of deoxyuridylate from dUTP in place of thymidylate during DNA synthesis. The deamination of cytosine can occur under physiological conditions (1,2) and would, of course, require correction in order to eliminate a resulting transition mutation. The incorporation of deoxyuridylate from dUTP might occur at a high frequency, but experimental evidence is indirect. First, dUTP is efficiently incorporated into DNA by DNA polymerase in vitro (3);

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“…Abasic sites, which are the most common lesions in the cell, are among the strongest of poisons [173,174,176,177,179]. Abasic sites are generated by a variety of methods, including spontaneous hydrolysis, DNA reactive chemicals, ionizing radiation, and base excision repair pathways [180][181][182]. It is estimated that some human cells may contain in excess of 100,000 abasic sites per genome, even in the absence of environmental stress [182].…”

Section: Dna Lesions As Topoisomerase II Poisonsmentioning

confidence: 99%

DNA topoisomerase II, genotoxicity, and cancer

McClendon

Osheroff

2007

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

363

466

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Type II topoisomerases are ubiquitous enzymes that play essential roles in a number of fundamental DNA processes. They regulate DNA under-and overwinding, and resolve knots and tangles in the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate segment of DNA. Because type II topoisomerases generate DNA strand breaks as a requisite intermediate in their catalytic cycle, they have the potential to fragment the genome every time they function. Thus, while these enzymes are essential to the survival of proliferating cells, they also have significant genotoxic effects. This latter aspect of type II topoisomerase has been exploited for the development of several classes of anticancer drugs that are widely employed for the clinical treatment of human malignancies. However, considerable evidence indicates that these enzymes also trigger specific leukemic chromosomal translocations. In light of the impact, both positive and negative, of type II topoisomerases on human cells, it is important to understand how these enzymes function and how their actions can destablize the genome. This article discusses both aspects of human type II topoisomerases.

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Heat-induced depyrimidination of deoxyribonucleic acid in neutral solution

Cited by 205 publications

References 20 publications

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

DNA topoisomerase II, genotoxicity, and cancer

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