Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence

Fundador, Erwin; Rusling, James F.

doi:10.1007/s00216-006-1041-x

Cited by 25 publications

(13 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many different techniques have been used to label, identify and quantify AP sites (7–14). It is difficult to use some of the techniques with a large number of samples because they either use equipment such as HPLC or use radioisotopes that are incompatible with a clinical setting.…”

Section: Introductionmentioning

confidence: 99%

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

et al. 2015

View full text Add to dashboard Cite

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

show abstract

Section: Introductionmentioning

confidence: 99%

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Below saturating MMS concentrations, the steady-state ligation rate is expected to equal the rate at which lesions are generated. Based on previous in vitro and in vivo reports (41,42), we estimated the damage rate by 25 mM MMS to ∼430-460 lesions·chromosome…”

Section: Mms (Simentioning

confidence: 99%

Single-molecule DNA repair in live bacteria

Uphoff

Reyes‐Lamothe

Leon

et al. 2013

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

183

283

View full text Add to dashboard Cite

Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.single-molecule tracking | super-resolution microscopy | DNA damage response | protein-DNA interaction | cytosolic diffusion A ll cellular organisms rely on complex DNA repair mechanisms for faithful chromosome replication and maintenance of their genome integrity (1). The variety of DNA lesions requires modular repair pathways that carry out damage recognition, damage removal, repair synthesis, and ligation in sequential steps catalyzed by a series of enzymes. However, all repair pathway steps need to be precisely balanced to avoid accumulation of DNA intermediates that are typically more mutagenic and toxic than the original lesion (2). Rapid processing of gapped and nicked intermediates is particularly crucial (3) because they provoke lethal double-strand breaks upon encountering replication forks (4); a single such break can lead to chromosome loss and cell death.Despite extensive genetic, biochemical, and biophysical studies (1), the molecular organization of DNA repair in vivo remains unclear. Most of our mechanistic understanding relies on in vitro ensemble studies, which cannot replicate the cellular environment and stochastic nature of chemical reactions. By avoiding ensembleaveraging, single-molecule experiments have revolutionized the study of protein-DNA interactions in vitro, but extension of these powerful concepts to DNA repair measurements in living cells remains an open goal. Early in vivo work focused on the mean behavior of cell populations and could not examine functionally important heterogeneity, such as the variation in protein copy numbers between cells and over time (5, 6). Such variation can lead to different repair rates across genetically identical cells and may dera...

show abstract

“…A report by Asaeda 33 and our previous paper 32 show that abasic sites are formed selectively when these alkylated bases are depurinated by heating.…”

Section: Optimization Of Depurination Conditionsmentioning

confidence: 78%

“…32 In the present paper, we report a CE-LIF array method to accurately size DNA fragments that result from cleavage at damaged bases. Like 32 P-PAGE, 20 this method can be used to determine the sequence specificity of DNA damage as long as cleavage can be done at the damage points.…”

mentioning

confidence: 99%

Accurate DNA Fragment Sizing by Capillary Electrophoresis with Laser-Induced Fluorescence Array for Detection of Sequence Specificity of DNA Damage

et al. 2008

View full text Add to dashboard Cite

Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo [a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a […GGCGCG-CAG…] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene.This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with 32 P end labeling.Cytochromes P450 in oxidative liver metabolism convert foreign hydrophobic substances into hydrophilic compounds that can be more readily excreted by the kidneys. 1 However, some metabolites may be chemically reactive electrophiles. These reactive metabolites may damage DNA, proteins, and other biomolecules or be inactivated by metabolic bioconjugation enzymes. 2 DNA damage often involves covalent binding of reactive metabolites to nucleobases, which may then lead to apoptosis (programmed cell death) or to neoplastic transformations.Cancer has been linked to specific changes within the amino acid sequence of certain proteins that control the movement of cells through the cell cycle, e.g., the p53 tumor suppressor protein. 3,4 These amino acid changes result from mutations in specific codons in the genes. The majority of human cancers are found to have mutations in the p53 gene. [5][6][7] These mutations do not occur at random within the gene but occur at specific positions with , adduct formation at the first guanine of codon 248 of the p53 gene causes G→T transversions. 3 This results in a point mutation in the p53 tumor suppressor protein, the substitution of an arginine for leucine at residue 248. The loss of the tumor suppressive capabilities of this mutated protein may contribute to uncontrolled cell growth or cancer. 11,12 Mapping the nucleobase sequence specificity for reaction of a reactive metabolite with DNA can be used as a predictive tool for carcinogenicity. There are two methods for detecting the sequence specificity of a DNA-damaging compound. The first involves isotopic 15 N or 13 C labeling of specific nucleobases within an oligonucleotide and determining the amount of damage at the site by mass spectrometry. [13][14][15][16] This method is rapid and also identifies the type of nucleobase adduct formed at the specific site. However, isotope-labele...

show abstract

Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence

Cited by 25 publications

References 32 publications

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

Single-molecule DNA repair in live bacteria

Accurate DNA Fragment Sizing by Capillary Electrophoresis with Laser-Induced Fluorescence Array for Detection of Sequence Specificity of DNA Damage

Contact Info

Product

Resources

About