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Title: Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

Abstract

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with (/sup 3/H)thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal,more » suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.« less

Authors:
; ; ;
Publication Date:
OSTI Identifier:
5120850
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 9
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; DNA REPAIR; BIOLOGICAL PATHWAYS; DNA; FIBROBLASTS; PHOTOREACTIVATION; PYRIMIDINE DIMERS; RADIOCHROMATOGRAPHY; SKIN; THYMIDINE; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ULTRAVIOLET RADIATION; XERODERMA PIGMENTOSUM; ANIMAL CELLS; AZINES; BIOLOGICAL RECOVERY; BIOLOGICAL REPAIR; BODY; CHROMATOGRAPHY; CONNECTIVE TISSUE CELLS; DISEASES; ELECTROMAGNETIC RADIATION; HETEROCYCLIC COMPOUNDS; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; NUCLEIC ACIDS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PYRIMIDINES; RADIATIONS; RECOVERY; REPAIR; RIBOSIDES; SEPARATION PROCESSES; SKIN DISEASES; SOMATIC CELLS; 560111* - Radiation Effects on Biochemicals- In Vitro- (-1987)

Citation Formats

Weinfeld, M, Gentner, N E, Johnson, L D, and Paterson, M C. Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts. United States: N. p., 1986. Web. doi:10.1021/bi00357a055.
Weinfeld, M, Gentner, N E, Johnson, L D, & Paterson, M C. Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts. United States. https://doi.org/10.1021/bi00357a055
Weinfeld, M, Gentner, N E, Johnson, L D, and Paterson, M C. 1986. "Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts". United States. https://doi.org/10.1021/bi00357a055.
@article{osti_5120850,
title = {Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts},
author = {Weinfeld, M and Gentner, N E and Johnson, L D and Paterson, M C},
abstractNote = {To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with (/sup 3/H)thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.},
doi = {10.1021/bi00357a055},
url = {https://www.osti.gov/biblio/5120850}, journal = {Biochemistry; (United States)},
number = ,
volume = 9,
place = {United States},
year = {Tue May 06 00:00:00 EDT 1986},
month = {Tue May 06 00:00:00 EDT 1986}
}