Processing of DNA base damage by DNA polymerases. Dihydrothymine and beta-ureidoisobutyric acid as models for instructive and noninstructive lesions.
The processing of unrepaired DNA lesions is a key to understanding and predicting the biological end points of particular DNA damages. In this study, we prepared single-stranded f1 phage (f1-K12) DNA containing dihydrothymine or beta-ureidoisobutyric acid as models for instructive or noninstructive base lesions and assessed the potential biological consequences of these lesions in vitro and in vivo. To determine the effect of the two lesions on in vitro DNA synthesis, the extent of DNA synthesis was measured by 3H-labeled nucleotide incorporation, and the newly synthesized DNA was analyzed by DNA sequencing gels. The results showed that dihydrothymine in the template was at most a weak block to in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (Pol I) and T4 DNA polymerase. In contrast, beta-ureidoisobutyric acid constituted a very strong (probably absolute) replicative block in vitro. With Pol I, termination bands were observed either opposite or one base prior to (3' to) the putative beta-ureidoisobutyric acid depending on its position in the template. However, when DNA synthesis was catalyzed by Pol I lacking a 3'----5' exonuclease activity, termination bands were only observed opposite beta-ureidoisobutyric acid, with purine nucleotides being incorporated preferentially opposite the lesion. With T4 DNA polymerase that contains a very active 3'----5' exonuclease activity, DNA synthesis was arrested almost exclusively one base prior to (3' to) the putative beta-ureidoisobutyric acid site in the template. We also measured survival of transfecting DNA containing dihydrothymine or beta-ureidoisobutyric acid in an attempt to correlate the in vitro data with in vivo processing. In keeping with the results obtained in vitro, dihydrothymine present in transfecting f1-K12 DNA did not constitute an inactivating lesion. On the other hand, it took 0.9 beta-ureidoisobutyric acid residues per molecule to inactivate transfecting f1-K12 DNA, indicating that the lesion was an absolute replicative block in vivo. When host cells were ultraviolet-irradiated to induce the SOS response, a slight increase (about 2-fold) in survival of transfecting f1-K12 DNA containing beta-ureidoisobutyric acid was observed. The potential effects of the structures of base lesions on lesion-polymerase interactions are discussed.
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Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405.
Petrullo
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Hatahet
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Wallace
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D001435:Bacteriophages; D001483:Base Sequence; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005609:Free Radicals; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011839:Radiation, Ionizing; D013604:T-Phages / Q000201:enzymology; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry; D014162:Transfection; D014508:Urea / Q000031:analogs & derivatives* / Q000737:chemistry
D005609:Free Radicals; C066926:beta-ureidoisobutyric acid; C042836:5,6-dihydrothymine; D014508:Urea; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D013941:Thymine