Abstract
The repair by UvrABC endonuclease of two major adducts formed by aflatoxin B1 in DNA was found to be similar. Aflatoxin epoxide was used to generate the aflatoxin B1.N7-guanine adduct which can convert to aflatoxin B1-formamidopyrimidine adduct. The reaction of the aflatoxin B1 epoxide with DNA follows pseudo-first order kinetics. The DNA sequence-specific relative reactivity of the epoxide is the same as previously observed for aflatoxin B1 activated by liver microsomes, therefore strongly reinforcing the notion that aflatoxin B1 reacts with DNA through the epoxide intermediate. For the majority of lesion sites, a high affinity protein-DNA complex was formed from the UvrA and the UvrB proteins with similar efficiency to both adducts, and to pyrimidine dimers, and then nicks the DNA when UvrC was added. The two incisions are at the eighth phosphodiester moiety 5' and the sixth phosphodiester moiety 3' of a modified guanine nucleotide. Both incisions appeared to be concerted. For some sites, the DNA sequence can alter the relative incision efficiency up to 15-fold. However, the majority of these AFB1 lesion structures in most DNA sequences are similar with respect to recognition by this nucleotide excision repair enzyme. Therefore the observation that the aflatoxin B1.N7-guanine lesion is removed rapidly, while the aflatoxin B1-formamidopyrimidine lesion persists in the mammalian cell may have other mechanistic explanations.
Highlights
RESULTS AND DISCUSSIONThe relative intensities of the bandswere apparently the same whether the labeling was from the 5' termini or from the 3' termini, except for the data in Fig. 6 for the 3'labeled bottom strand where over-representation of the side, the above analyses of supercoiled DNA are important, shorter bandsappeared to have resulted
From the 4Znstitute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, the TDiuision of Toxicologyand Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the IISchool of Hygiene and Public Health, The Johns HopkinsUniversity, Baltimore, Maryland 29001
Its chemical reactivity is thought to aflatoxin B1*iV"guanineadduct which can convert to proceed via the formation of the 8,9 epoxide (Fig. 1) that can aflatoxin B1-formamidopyrimidine adduct
Summary
The relative intensities of the bandswere apparently the same whether the labeling was from the 5' termini or from the 3' termini, except for the data in Fig. 6 for the 3'labeled bottom strand where over-representation of the side, the above analyses of supercoiled DNA are important, shorter bandsappeared to have resulted This bias may have because superhelicity is known to have large effects on the arisen from a relatively higher concentration of AFBl lesions efficiencies of UvrABC endonuclease repair of lesions of an- such that some molecules were nicked more than once.
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