Comparison of the efficiency of synthesis past single bulky DNA adducts in vivo and in vitro by the polymerase III holoenzyme.
Previous studies from our laboratory revealed that site-specific and stereospecific styrene oxide (SO) lesions in M13 DNA were readily bypassed when transfected into Escherichia coli cells, but these same lesions blocked the progress of several purified polymerases in vitro when situated in oligodeoxynucleotide templates (Latham, G. J., et al. (1993) J. Biol. Chem. 268, 23427-23434; Latham, G. J., et al. (1995) Chem. Res. Toxicol. 8, 422-430). To resolve this apparent discrepancy, we constructed single-stranded M13 genomes containing single SO adducts and compared their replication efficiencies in E. coli cells to the extent of bypass synthesis in vitro using three different complexes of the purified E. coli polymerase III (Pol III) holoenzyme. The transformation efficiencies of the SO-adducted M13 templates were comparable to those of the nonadducted controls, indicating facile bypass in E. coli. When the identical adducted M13 vectors were replicated in vitro with the reconstituted complexes of the Pol III holoenzyme, the results were consistent with the in vivo data: Synthesis past two of the three SO adducts in M13 was unhindered relative to synthesis on the unadducted M13 control template. Since our previous in vitro assays indicated that SO adducts in 33-mer templates largely blocked polymerases other than Pol III, we repeated these studies using reconstituted Pol III. Significantly, Pol III replication was poorly processive and strongly terminated by SO lesions in 33-mer templates. This result was in stark contrast to the efficient bypass in vitro of the same adducts in M13 DNA. In fact, Pol III-mediated bypass was enhanced to > 75-fold on adducted circular M13 templates as compared to adducted linear oligodeoxynucleotides. The implications of the effects of polymerase processivity and template-primer structure upon lesion bypass are discussed.
Latham
G J
GJ
Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232, USA.
McNees
A G
AG
De Corte
B
B
Harris
C M
CM
Harris
T M
TM
O'Donnell
M
M
Lloyd
R S
RS
D017104:Bacteriophage M13 / Q000378:metabolism; D002478:Cells, Cultured; D003067:Coenzymes / Q000378:metabolism* / Q000494:pharmacology; D018736:DNA Adducts / Q000009:adverse effects* / Q000032:analysis*; D004258:DNA Polymerase III / Q000378:metabolism* / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*
D003067:Coenzymes; D018736:DNA Adducts; D004258:DNA Polymerase III