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PMID: 9461450 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Nucleic acids research ·Vol. 26 ·No. 4 ·1998-02-15 ·Pages 932-41

Harrison L, Hatahet Z, Purmal AA, Wallace SS

Abstract

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.

MeSH Terms
Base Sequence Binding Sites DNA Damage DNA Repair Deoxyribonuclease (Pyrimidine Dimer) Endodeoxyribonucleases/metabolism Escherichia coli/enzymology Escherichia coli Proteins Free Radicals/toxicity Molecular Sequence Data Oligodeoxyribonucleotides/chemistry,genetics,metabolism Oxidation-Reduction Purines/chemistry Pyrimidines/chemistry Substrate Specificity
Chemicals
Escherichia coli Proteins Free Radicals Oligodeoxyribonucleotides Purines Pyrimidines Endodeoxyribonucleases Deoxyribonuclease (Pyrimidine Dimer) NTH protein, E coli
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Harrison L
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.
Hatahet Z
Purmal A A
Wallace S S
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/26.4.932
Published
1998-02-15
Pages
932-41
Language
English
Region
England
NLM ID
0411011
PMCID
PMC147348
Subset
IM
Grants
NCI NIH HHS · R37CA33657 · United States
External Links
PubMed source DOI: 10.1093/nar/26.4.932 PMC Full Text View journal details
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