Despite recent experiments showing that BrdUrd-induced mutagenesis can be independent of the level of bromouracil (BrUra) 5-Bromouracil (BrUra), an analogue of thymine, and 2-aminopurine (APur), an analogue of adenine, cause transition mutations in various organisms (1-14). A-T--'G-C and G-C oA-T transitions are induced by both base analogues; however, the extent of their bidirectionality remains unclear (8-21). It is known that BrUra and APur are incorporated "normally" into DNA opposite adenine and thymine, respectively. Ambiguities in their base pairing properties (BrUra can pair with guanine and APur with cytosine) are clearly important determinants in their mutagenicity and lead to what Freese (9) and Rudner (19) defined as replication errors and incorporation errors.Under certain conditions, BrUra substitution in DNA is not correlated with BrdUrd mutagenesis. This point was forcefully made in recent experiments by Kaufman and Davidson (22) using Syrian hamster melanoma cells and by Aebersold using Chinese hamster cells (1). For example, in the Kaufman and Davidson experiment it was shown that BrdUrd-induced mutation frequencies in a Syrian hamster melanoma cell line depended not on how much BrUra was stably substituted for thymine in the DNA but rather on the concentration of BrdUrd in the cell growth medium. In view of the evidence in Salmonella (14) and in bacteriophage T4 (23), which supports the idea that BrUra increases replication errors when acting in a template capacity, the Syrian hamster melanoma results are remarkable, to say the least.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1801It will be of fundamental importance in understanding the mechanism of BrdUrd mutagenesis to recognize that the uncoupling of the level of BrUra substitution in DNA from the level of BrdUrd mutagenesis was achieved through the addition of deoxythymidine or deoxycytidine to the BrdUrd mutagenesis medium (1, 2, 22). The addition of deoxythymidine resulted in less, or even undetectable, BrUra substitution in DNA but the same, or increased, level of mutagenesis (1, 2, 22). Conversely, the addition of deoxycytidine with BrdUrd during mutagenesis resulted in a complete inhibition of mutagenesis but full substitution of BrUra for thymine in DNA (2).These observations have led some investigators to conclude that BrdUrd mutagenesis occurs because of an altered physiological state such as."deoxycytidinelessness" (24) or to implicate BrdUrd metabolites as allosteric effectors of the fidelity of DNA synthetic enzymes (1). It has also been proposed that BrdUrd mutagenesis does not involve mispairing of BrUra during DNA synthesis (5, 10, 22, 25).The conclusion that BrUra base pairs exactly as thymine conflicts with certain experiments termed "clean growth" (23) in which BrUra-substituted DNA was proved to induce replication errors in the absence of exogeno...

We measured the in vivo incororation of 2-aminopurine into DNA of T4 bacteriophage allelic for gene 43 (DNA polymerase), mutator (L56), 43+, and antimutator (L141). The magnitude of incorporation (mol/mol of Thy) was 1/1500 in L56, 1/1600 in 43+, and 1/8900 in L141. The incorporation ratio L56:43+:L141 in vivo was equal to that mediated by the purified DNA polymerases of these allelic phages in vitro. A model for 2-aminopurine-induced A*T ;-GC transitions is discussed. The model is used to predict the magnitudes of replication errors (C mispairing with a template 2-aminopurine) and incorporation errors (2-aminopurine mispairing with a template C) per round of replication and to investigate the asymmetry in 2-aminopurine-induced transitions favoring the A*T --GC pathway over G-C -A-T. We suggest that the fidelity of 156 and L141 DNA pol1erases exemplifies one-step and two-step editing, respectively. Error detection and correction is a process basic to the maintenance of informational integrity in biological systems. Genetic studies (1-7) demonstrated that certain temperature-sensitive bacteriophage T4 mutants (mutators and antimutators) having single base substitutions in gene 43, the gene coding for DNA polymerase (8), can exhibit increased and decreased mutation frequencies, respectively, as measured by the revision of marker loci. Measurements of the insertion and removal of 2-aminopurine (AP) deoxynucleotides by purified mutator and antimutator DNA polymerases during in vitro DNA synthesis revealed that the fidelity of DNA replication can be influenced by the polymerase-associated 3'-exonuclease (9, 10) or proofreading activity (11). Similar studies on the mispairing of normal nucleotides using defined polymer templates (12,13) further demonstrated that the insertion specificity may also influence the fidelity of the polymerization process.AP, a base analogue of adenine, induces transition mutations through the pathway: A-T >± AP-T ;=t AP-C ;=t G-C (14). In this paper, we address experimentally the initial and final steps in AP-induced A-T --G-C transitions. The first step is measured as AP incorporation into encapsulated DNA in T4 mutator (L56), wild-type (43+), and antimutator (L141) backgrounds. The final step is measured as the AP-induced reversion (A-T G-C transition) of the marker locus ri UV199 in gene 43 allelic bacteriophage (5,6). Both AP incorporation and APinduced transitions vary in a manner consistent with L56 mutator or L141 antimutator phenotypes. We will show that mutator and antimutator phage differ much more widely in AP-induced marker reversion than in AP incorporation. Because they respond differently to AP mutagenesis, the T4 phage must progress through the stages of AP-induced mutagenesis at significantly different rates. Therefore, it is possible to gain an intimate view of AP-induced mutagenesis and the role of A model of AP-induced transitions is proposed that facilitates analysis of the data by relating the population of each possible base pair in the transition pathway to ...

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