Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein

Karakaya, Asuman

doi:10.1093/nar/25.3.474

Cited by 162 publications

(129 citation statements)

References 36 publications

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“…Previous studies employing conventional assays have provided kinetic parameters for Fpg-catalyzed 8-oxodG base excision/ elimination, thereby furnishing insight into the origins of Fpg catalytic specificity (3,22,23). However, the data reported differ substantially because of variability in the experimental conditions used (13).…”

Section: Advantages Of the Cha Methods And Comparisons With Published mentioning

confidence: 99%

A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision

Minetti

Remeta

Breslauer³

2008

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

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We report a continuous hyperchromicity assay (CHA) for monitoring and characterizing enzyme activities associated with DNA processing. We use this assay to determine kinetic and thermodynamic parameters for a repair enzyme that targets nucleic acid substrates containing a specific base lesion. This optically based kinetics assay exploits the free-energy differences between a lesion-containing DNA duplex substrate and the enzymecatalyzed, lesion-excised product, which contains at least one hydrolyzed phosphodiester bond. We apply the assay to the bifunctional formamidopyrimidine glycosylase (Fpg) repair enzyme (E) that recognizes an 8-oxodG lesion within a 13-mer duplex substrate (S). Base excision/elimination yields a gapped duplex product (P) that dissociates to produce the diagnostic hyperchromicity signal. Analysis of the kinetic data at 25°C yields a K m of 46.6 nM for the E⅐S interaction, and a k cat of 1.65 min ؊1 for conversion of the ES complex into P. The temperature dependence reveals a free energy (⌬G b) of ؊10.0 kcal⅐mol ؊1 for the binding step (E ؉ S 7 ES) that is enthalpy-driven (⌬H b ‫؍‬ ؊16.4 kcal⅐mol ؊1 ). The activation barrier (⌬G ‡ ) of 19.6 kcal⅐mol ؊1 for the chemical step (ES 7 P) also is enthalpic in nature (⌬H ‡ ‫؍‬ 19.2 kcal⅐mol ؊1 ). Formation of the transition state complex from the reactants (E ؉ S 7 ES ‡ ), a pathway that reflects Fpg catalytic specificity (k cat/Km) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (⌬G T ‡ ) of 9.6 kcal⅐mol ؊1 . These parameters characterize the driving forces that dictate Fpg enzyme efficiency and specificity and elucidate the energy landscape for lesion recognition and repair.8-oxoguanosine ͉ activation energy ͉ energetics ͉ glycosylase/lyase T he number of DNA lesions implicated in genetic diseases and degenerative cell disorders has increased over the past decades (1, 2). Such findings have stimulated efforts to characterize specific enzyme families that function in the recognition and repair of DNA damage (3-7). The majority of existing methods used to determine kinetic parameters for base/nucleotide excision repair (BER/NER) enzymes employ labeling and quenching techniques that measure either product formation or substrate disappearance (3,(8)(9)(10)(11). Although of great value, many of these assays require chemical modifications and/or sampling of the enzyme-catalyzed reaction at discrete time intervals. These features may potentially compromise successful monitoring of intrinsic system properties. To address this challenge, we have developed a continuous hyperchromicity assay (CHA) that exploits the intrinsic free-energy differences of a damaged DNA duplex substrate and the resultant processed product. The method requires minimal sample manipulation and provides a real-time, continuous optical signal to quantify product formation. Because the presence of a damaged base or lesion often exerts profound effects on DNA duplex energetics and stability (12), the solution conditions of the assay are optimized to en...

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Section: Advantages Of the Cha Methods And Comparisons With Published mentioning

confidence: 99%

A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision

Minetti

Remeta

Breslauer³

2008

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

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“…19), but some evidence suggests that repair of 8-OH-Ade is mechanistically different from repair of 8-OHGua. Among the bacterial and mammalian DNA glycosylases, which were investigated for their activity on lesions in oxidatively damaged DNA, only Escherichia coli formamidopyrimidine glycosylase (Fpg) exhibited a low activity on 8-OH-Ade in DNA containing multiple modified bases (35,36). However, this activity was insignificant when compared with the activity of Fpg on 8-OH-Gua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), and 4,6-diamino-5-formamidopyrimidine (FapyAde), which are the principal substrates of Fpg.…”

Section: Fig 2 Structure Of Csb and The Location Of Designed Mutantsmentioning

confidence: 99%

The Cockayne Syndrome Group B Gene Product Is Involved in Cellular Repair of 8-Hydroxyadenine in DNA

Tuo¹,

Jaruga²,

Rodriguez³

et al. 2002

Journal of Biological Chemistry

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Cockayne syndrome (CS) is a human disease characterized by sensitivity to sunlight, severe neurological abnormalities, and accelerated aging. CS has two complementation groups, CS-A and CS-B. The CSB gene encodes the CSB protein with 1493 amino acids. We previously reported that the CSB protein is involved in cellular repair of 8-hydroxyguanine, an abundant lesion in oxidatively damaged DNA and that the putative helicase motif V/VI of the CSB may play a role in this process. The present study investigated the role of the CSB protein in cellular repair of 8-hydroxyadenine (8-OHAde), another abundant lesion in oxidatively damaged DNA. Extracts of CS-B-null cells and mutant cells with site-directed mutation in the motif VI of the putative helicase domain incised 8-hydroxyadenine in vitro less efficiently than wild type cells. Furthermore, CS-B-null and motif VI mutant cells accumulated more 8-hydroxyadenine in their genomic DNA than wild type cells after exposure to ␥-radiation at doses of 2 or 5 Gy. These results suggest that the CSB protein contributes to cellular repair of 8-OH-Ade and that the motif VI of the putative helicase domain of CSB is required for this activity.

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“…To this end, we were motivated to develop a sensitive, robust and general approach to quantify DNA lesions caused by the known classes of inflammation chemistry shown in Figure 2. It should be noted that there are many different products of DNA oxidation and alkylation arising from exposure to ionizing radiation and other insults, [18][19][20][21][22][23][24] but these species are not unlikely to form under the biological conditions of inflammation. 4 Further, we have recently initiated efforts to include the cytosine and guanine halo-adducts arising from reactions of DNA with neutrophil-derived HOCl.…”

Section: Introductionmentioning

confidence: 99%

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

et al. 2008

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The analysis of damage products as biomarkers of inflammation has been hampered by a poor understanding of the chemical biology of inflammation, the lack of sensitive analytical methods, and a focus on single chemicals as surrogates for inflammation. To overcome these problems, we developed a general and sensitive liquid chromatographic tandem mass spectrometry (LC/MS-MS) method to quantify, in a single DNA sample, the nucleoside forms of seven DNA lesions reflecting the range of chemistries associated with inflammation: 2′-deoxyuridine, 2′-deoxyxanthosine, and 2′-deoxyinosine from nitrosative deamination; 8-oxo-2′-deoxyguanosine from oxidation; and 1,N 2 -etheno-2′-deoxyguanosine, 1,N 6 -etheno-2′-deoxyadenosine, and 3,N 4 -etheno-2′-deoxycytidine arising from reaction of DNA with lipid peroxidation products. Using DNA purified from cells or tissues under conditions that minimize artifacts, individual nucleosides are purified by HPLC and quantified by isotope-dilution, electrospray ionization LC/MS-MS. The method can be applied to other DNA damage products and requires 4-6 days to complete depending upon the number of samples. Search terms

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Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein

Cited by 162 publications

References 36 publications

A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision

A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision

The Cockayne Syndrome Group B Gene Product Is Involved in Cellular Repair of 8-Hydroxyadenine in DNA

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

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