DNA nick processing by exonuclease and polymerase activities of bacteriophage T 4 DNA polymerase accounts for acridine - induced mutation specificities in T 4 ( frameshift / mutagenesis / 9 - aminoacridine / T 4

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3' -* 5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5' ->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridineinduced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage. Initial studies of protein sequence changes produced by spontaneous and acridine-induced frameshift mutations in the lysozyme gene of bacteriophage T4 revealed that single base deletions or duplications frequently occurred within A-T runs (1). It was therefore proposed that both deletions and duplications occur because one DNA strand could stably misalign upon the complementary strand within the base run in two ways: a bulged template strand produces a deletion in the elongating DNA, whereas a bulged elongated strand produces a duplication (1). Acridine-induced mutations generally consistent with the predictions of this slippage model were observed in the Escherichia coli lacI gene (2, 3) and in bacteriophage A (4). In these systems, the induced frameshifts occur preferentially in G-C runs. Subsequent DNA sequencing of acridine-induced mutants in the T4 lysozyme gene identified frequent frameshifts that were neither within nor adjacent to base runs or other repeats and therefore could not be explained by DNA strand slippage (5, 6). The slippage model also fails to explain the specificities of many acridine-induced frameshift mutations that arise at hotspot sites in the rIIB and thymidylate synthase genes of T4 (7, 8). For example, a frequent mutation within the rIIB sequence 5'-AAATTGTTAAACT-3' is the duplication of the G. Because this G is flanked on both sides by two T residues, the G cannot slip to form a complementary base pair. Moreover, despite the high frequency ofG duplications, G deletions are not induced as would be predicted by slippage. The specificities of deletions and duplications at the rIIB hotspot site are consistent with a different model. In this model, bases are deleted or duplicated by exonuclease or by DNA polymerase, respectively, at the 3' ends of acridine-induced, T4 topoisomerase-generated nicks (Fig. 1) (9). The frameshift mutations are created by religation of the shortened or extended 3' end to the original 5' end of the nick (10). In vitro, the products of acridine-induced, topoisomerase-generated nicks are a free 3'-hydroxyl end and a 5'-phosphate end that remains covalently linked to the topoisomerase (11). The sites at which this in vitro reaction occur in T4 DNA correlate with the preferred sites of in vivo mutations (7, 10, 12). In contrast to the slippage model, this model accounts entirely for the specificity of mutations arising at the T4 rIIB hotspot (7, 10). For example, this nick-processing model, coupled with the observed sites of acridine-induced cleavage, predicts G duplications and accounts for the absence of G deletions discussed above. Alternative processing at the same nick predicts deletion, but not the duplication, of the T 5' to the G. Consistent with this prediction, deletions, but not duplications, are frequently induced. Moreover, the preponderance of deletions is inconsistent with the predictions of slippage, which predicts duplications. The surrounding DNA sequence provides further evidence that A-run sites are not automatic hotspots for acridine-induced frameshifts. The Arun upstream of the G is not cleaved in vitro and mutations are not increased by acridines; the A run downstream of the G is cleaved and mutations are increased by acridines (10). In support of the proposed role of the T4 topoisomerase in acridine mutagenesis: (i) mutagenesis is abolished in acridinetreated T4 phage having an amber mutation in one subunit of the topoisomerase (10); (ii) the in vitro acridine-induced topoisomerase cleavage sites correspond precisely to the positions of in vivo acridine-induced frameshift sites (7, 10); and (iii) when new topoisomerase cleavage sites are created by introducing DNA sequence changes in the vicinity of the rIIB hotspot, acridineinduced frameshifts arise at these new sites with exactly the specificities predicted by the nick-processing model (12). Because T4 DNA polymerase contains both a 3' exonuclease and polymerase activities, this enzyme alone might account Abbreviation: 9-AA, 9-aminoacridine. *To whom reprint requests should be addressed at: Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103. 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