Abstract
Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.
Highlights
Recent studies suggest that in addition to DNA polymerases1 I–III, which are prototypes of the A, B, and C-polymerase families, respectively [1, 2], Escherichia coli possesses two members of the recently described Y-family of DNA polymerases [3]
Contact between pol V and the 3Ј-tip of the RecA nucleoprotein filament is required both for translesion synthesis (TLS) to occur and for pol V to remain bound at a primer 3Ј-end (39 – 41)
Of the filament in the 5Ј to 3Ј direction occurred in the presence of ATP, but not ATP␥S [42, 43], causing termination of pol V synthesis roughly 1– 4 bases downstream from the lesion (Fig. 2, upper left panel, lane 5 for both the S and R isomers). pol V showed a somewhat greater TLS efficiency when replicating beyond the R rather than the S isomer (Table I, ATP␥S column)
Summary
Materials—Ultrapure ATP, dNTPs, E. coli single-stranded DNAbinding protein (SSB), and RecA were purchased from Amersham Biosciences, Inc. Three sequence contexts (one for A and two for G adducts) that had previously been used to examine the mutational consequences of these adducts in an E. coli M13 system were used [30] For each of these adducts, two diastereomers are possible, corresponding to an S or R configuration at the point of attachment of the hydrocarbon to the purine base (see Fig. 1A); and both isomers, the BaP DE-S and -R adducts, respectively, were examined. Linear BaP DE-containing DNA templates used in the lesion bypass assay and fidelity study were prepared by ligation of linearized single-stranded M13mp DNA with BaP DE-containing oligonucleotides and a synthetic 48-mer as previously described [35]. The reaction conditions were the same as in the translesion synthesis assay, except that different sets of primers were annealed to the template, and incorporation of individual dNTP substrates was measured as a function of concentration to determine apparent Vmax and Km values for each substrate. Analysis of progeny bacteriophage was carried out by a combination of probe hybridization and sequencing following the protocol referred to as “Experiment I” in Ref. 30
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