32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase.

2.50
Hdl Handle:
http://hdl.handle.net/10541/104601
Title:
32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase.
Authors:
Povey, Andrew C; Cooper, Donald P; Littler, Edward
Abstract:
Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.
Affiliation:
Cancer Research Campaign Department of Carcinogenesis, Patterson Institute for Cancer Research, Manchester, UK.
Citation:
32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase. 1991, 12 (4):709-12 Carcinogenesis
Journal:
Carcinogenesis
Issue Date:
Apr-1991
URI:
http://hdl.handle.net/10541/104601
DOI:
10.1093/carcin/12.4.709
PubMed ID:
1849471
Type:
Article
Language:
en
ISSN:
0143-3334
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorPovey, Andrew Cen
dc.contributor.authorCooper, Donald Pen
dc.contributor.authorLittler, Edwarden
dc.date.accessioned2010-06-10T09:00:20Z-
dc.date.available2010-06-10T09:00:20Z-
dc.date.issued1991-04-
dc.identifier.citation32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase. 1991, 12 (4):709-12 Carcinogenesisen
dc.identifier.issn0143-3334-
dc.identifier.pmid1849471-
dc.identifier.doi10.1093/carcin/12.4.709-
dc.identifier.urihttp://hdl.handle.net/10541/104601-
dc.description.abstractAlkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.en
dc.language.isoenen
dc.subject.meshAdenosine Triphosphate-
dc.subject.meshAlkylation-
dc.subject.meshAutoradiography-
dc.subject.meshChromatography, Thin Layer-
dc.subject.meshHerpesvirus 4, Human-
dc.subject.meshHydrogen-Ion Concentration-
dc.subject.meshKinetics-
dc.subject.meshNucleosides-
dc.subject.meshPhosphorus Radioisotopes-
dc.subject.meshPhosphorylation-
dc.subject.meshThymidine-
dc.subject.meshThymidine Kinase-
dc.title32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase.en
dc.typeArticleen
dc.contributor.departmentCancer Research Campaign Department of Carcinogenesis, Patterson Institute for Cancer Research, Manchester, UK.en
dc.identifier.journalCarcinogenesisen

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