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PMID: 1549115 Published · ppublish English Journal Article

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

Molecular and cellular biology ·Vol. 12 ·No. 4 ·1992-04-00 ·Pages 1605-12

Dianov G, Price A, Lindahl T

Abstract

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.

MeSH Terms
Apurinic Acid/metabolism Base Sequence Cell Extracts Cell Line/metabolism DNA Polymerase I/antagonists & inhibitors DNA Repair DNA Replication Deoxyribonucleases, Type II Site-Specific/metabolism Escherichia coli/metabolism Humans Models, Genetic Molecular Sequence Data Oligodeoxyribonucleotides/metabolism Uracil/metabolism
Chemicals
Cell Extracts Oligodeoxyribonucleotides Apurinic Acid Uracil DNA Polymerase I Deoxyribonucleases, Type II Site-Specific
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Dianov G
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom.
Price A
Lindahl T
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.12.4.1605-1612.1992
Published
1992-04-00
Pages
1605-12
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC369603
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.12.4.1605-1612.1992 PMC Full Text View journal details
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