We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)

The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174 RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, we have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174 RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, we have found that UVRABC nuclease incises at the eighth phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand.(ABSTRACT TRUNCATED AT 250 WORDS)

N-(Deoxyguanosin-C8-yl)-2-(acetylamino)fluorene (dG-C8-AAF) and N-(deoxyguanosin-C8-yl)-2-aminofluorene (dG-C8-AF) are the two major DNA adducts induced by the chemical carcinogen 2-(acetylamino)fluorene (AAF). Molecular modeling shows that, in the DNA double helix, dG-C8-AF can maintain an anti-structure and normal base pairing, while dG-C8-AAF favors a syn-structure and causes base displacement. In the phi X174 RF DNA-Escherichia coli transfection system, it has been found that dG-C8-AF is 7-10-fold less lethal than dG-C8-AAF; these results suggest that these two kinds of DNA adducts may have different effects on DNA replication and that they may be repaired by different pathways. We have investigated the effects of these two kinds of adducts on DNA polymerase III holoenzyme (pol III-H) and DNA polymerase I Klenow fragment (pol I-Kf) mediated DNA synthesis by using carcinogen-modified M13 single-stranded DNA hybridized with 32P-labeled primer as templates. We have found that pol III-H and pol I-Kf replicate through dG-C8-AF with 92% and 62% frequency, respectively; in contrast, these two enzymes replicate through dG-C8-AAF with only 38% and 25% frequency, respectively. AF-adducted DNA shows a more profound sequence specificity in blocking DNA synthesis than AAF-adducted DNA, and the sequence specificities in blocking DNA synthesis for both kinds of adducts differ for pol III-H and pol I-Kf.

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