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 s tereospecific styrene oxide (SO) lesions in M13 DNA were readily bypas sed when transfected into Escherichia coli cells, but these same lesio ns blocked the progress of several purified polymerases in vitro when situated in oligodeoxynucleotide templates (Latham, G. J., et al. (199 3) J. Biol. Chem. 268, 23427-23434; Latham, G. J., et al. (1995) Chem. Res. Toxicol, 8, 422-430). To resolve this apparent discrepancy, we c onstructed single-stranded M13 genomes containing single SO adducts an d compared their replication efficiencies in E. coli cells to the exte nt of bypass synthesis in vitro using three different complexes of the purified E. coli polymerase III (Pol III) holoenzyme. The transformat ion efficiencies of the SO-adducted M13 templates were comparable to t hose 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 we re consistent with the in, vivo data: Synthesis past two of the three SO adducts in M13 was unhindered relative to synthesis on the unadduct ed 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. S ignificantly, Pol III replication was poorly processive and strongly t erminated 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 D NA. In fact, Pol III-mediated bypass was enhanced to >75-fold on adduc ted circular M13 templates as compared to adducted linear oligodeoxynu cleotides. The implications of the effects of polymerase processivity and template primer structure upon lesion bypass are discussed.