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1.
PLoS One ; 11(6): e0158581, 2016.
Article in English | MEDLINE | ID: mdl-27362559

ABSTRACT

Mammalian cells are constantly and unavoidably exposed to DNA damage from endogenous and exogenous sources, frequently to the detriment of genomic integrity and biological function. Cells acquire a large number of chemically diverse lesions per day, and each can have a different genetic fate and biological consequences. However, our knowledge of how and when specific lesions are repaired or how they may compromise the fidelity of DNA replication or transcription and lead to deleterious biological endpoints in mammalian cells is limited. Studying individual lesions requires technically challenging approaches for the targeted introduction of defined lesions into relevant DNA sequences of interest. Here, we present a systematic analysis of factors influencing yield and an improved, efficient and reliable protocol for the production of mammalian expression phagemid vectors containing defined DNA base modifications in any sequence position of either complementary DNA strand. We applied our improved protocol to study the transcriptional mutagenesis-mediated phenotypic consequences of the common oxidative lesion 5-hydroxyuracil, placed in the G12 mutational hotspot of the KRAS oncogene. 5-OHU induced sustained oncogenic signaling in Neil1-/-Neil2-/- mouse cells. The resulting advance in technology will have broad applicability for investigation of single lesion DNA repair, mutagenesis, and DNA damage responses in mammalian cells.


Subject(s)
DNA Damage , DNA Repair , DNA/genetics , Genetic Vectors , Mutagenesis , Animals , Mutation
2.
Int J Biochem Cell Biol ; 37(6): 1331-6, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15778095

ABSTRACT

Genome integrity is maintained via removal (repair) of DNA lesions and an increased load of such DNA damage has been linked to numerous pathological conditions, including carcinogenesis and ageing. 8-Oxo-7,8-dihydroguanine is one of the most critical lesions of this type. The free 8-oxo-7,8-dihydroguanine produced by the action of a specific DNA glycosylase is a potential source of this compound in urine. To date, there has been no direct, experimental evidence demonstrating that urinary 8-oxo-7,8-dihydroguanine is produced by the base excision repair pathway. For clarification of this issue, we applied a recently developed methodology which involved high performance liquid chromatography pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection to compare the urinary excretion rate of 8-oxo-7,8-dihydroguanine in wild type and OGG1 glycosylase knock out mice. Our study revealed a 26% reduction in urinary level of 8-oxo-7,8-dihydroguanine in OGG1 deficient mice in comparison with the wild type strain. This clearly indicates that the mouse OGG1 glycosylase contributes significantly to the generation of urinary 8-oxo-7,8-dihydroguanine. Therefore, urinary measurements of 8-oxo-7,8-dihydroguanine may be attributed to DNA damage and repair, which in turn suggests that they may be useful in studying associations between DNA repair and disease.


Subject(s)
DNA Glycosylases/physiology , DNA Repair , Guanine/analogs & derivatives , Animals , Chromatography, High Pressure Liquid , DNA Damage/physiology , DNA Glycosylases/deficiency , Gas Chromatography-Mass Spectrometry , Guanine/urine , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
3.
Free Radic Res ; 38(11): 1201-5, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15621697

ABSTRACT

In order to eliminate the possibility that diet may influence urinary oxidative DNA lesion levels, in our experiments we used a recently developed technique involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection. This methodology was applied for the determination of the lesions: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-(hydroxymethyl)uracil (5HMUra) in the urine of mice fed with nucleic acid free diet and normal, unrestricted diet. The mean levels of 8-oxoGua, 8-oxodGuo and 5HMUra of the animals fed the normal diet reached the mean values of 15.6 +/- 3.5, 2.0 +/- 0.53 and 16.8 +/- 10.4 nmol/kg/24 h, After feeding the mice for 12 days with nucleic acid free diet the respective values were 18.8 +/- 4.6, 1.6 +/- 0.3 and 25.4 +/- 10.5 nmol/kg/24 h, respectively. The results clearly demonstrate that irrespective of the diet, the excretion rates were not statistically different during the course of feeding. The respective p values for the differences between lesions in the two types of diets were: 0.13 (8-oxoGua), 0.16 (8-oxodGuo), 0.18 (5-HMUra). Our results clearly indicate that diet does not contribute to urinary excretion of the lesions in mouse model.


Subject(s)
DNA Damage/drug effects , DNA/urine , Deoxyguanosine/analogs & derivatives , Diet , Guanine/analogs & derivatives , Oxidative Stress/drug effects , Pentoxyl/analogs & derivatives , 8-Hydroxy-2'-Deoxyguanosine , Animal Feed , Animals , Deoxyguanosine/urine , Guanine/urine , Humans , Male , Mice , Mice, Inbred C57BL , Oxidation-Reduction/drug effects , Pentoxyl/urine
4.
Mol Cell Biol ; 23(15): 5346-53, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12861020

ABSTRACT

Flap endonuclease 1 (FEN1) has been shown to remove 5' overhanging flap intermediates during base excision repair and to process the 5' ends of Okazaki fragments during lagging-strand DNA replication in vitro. To assess the in vivo role of the mammalian enzyme in repair and replication, we used a gene-targeting approach to generate mice lacking a functional Fen1 gene. Heterozygote animals appear normal, whereas complete depletion of FEN1 causes early embryonic lethality. Fen1(-/-) blastocysts fail to form inner cell mass during cellular outgrowth, and a complete inactivation of DNA synthesis in giant cells of blastocyst outgrowth was observed. Exposure of Fen1(-/-) blastocysts to gamma radiation caused extensive apoptosis, implying an essential role for FEN1 in the repair of radiation-induced DNA damage in vivo. Our data thus provide in vivo evidence for an essential function of FEN1 in DNA repair, as well as in DNA replication.


Subject(s)
Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/physiology , Alleles , Animals , Apoptosis , Blastocyst/metabolism , Cell Division , DNA Repair , DNA Replication , Flap Endonucleases , Gamma Rays , Genetic Vectors , Genotype , Heterozygote , Homozygote , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Microscopy, Fluorescence , Models, Genetic , Mutation , Oxidative Stress , Physical Chromosome Mapping , Polymerase Chain Reaction , Time Factors
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