2-Aminopurine-induced mutagenesis in T4 bacteriophage: a model relating mutation frequency to 2-aminopurine incorporation in DNA.

Abstract

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 ...

In vivo studies of repair of 2-aminopurine in Escherichia coli

“…3) was purified by isopycnic density centrifugation in cesium trifluoroacetate, digested with pancreatic DNase I and snake venom phosphodiesterase, and analyzed for [6-3H]deoxy-2-aminopurine monophosphate ([3H]d2APMP) ( Table 3). As has been reported previously (12,43) (11), more recent experiments question this interpretation since all of the surviving colonies were Mut- (31). Thus, the effect of 2AP on the mutation frequency of wild-type, dam mutH, and mutH strains was studied.…”

“…The data that least support the model for mismatch correction in the repair of 2AP:C mismatches are from experiments that quantitate the amount of 2AP that is incorporated into the DNA of T4-infected cells. According to these experiments only one 2AP is incorporated for every 6,400 bases (12). Of these only 5% are 2AP-C mismatches (51).…”

Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells.

“…Nor was the AraC-6-1 cell line generally impaired in its ability to repair DNA, as evidenced by its normal sensitivity to mutagenesis by the alkylating agent MNNG. We reasoned that, as predicted by one hypothesis (1,12,14,27), the perturbed TTP and dCTP pools were responsible for the increased mutability of AraC-61 cells by Apur. This was verified by pharmacologically manipulating the dCTP and TTP pools by adding thymidine to the growth medium.…”

“…In procaryotes, Apur is usually incorporated into DNA opposite T and induces A:T --G:C transitions by mispairing with C in a subsequent round of DNA replication. Apur can also induce G:C --A:T transitions, although with a much lower frequency, by being erroneously incorporated into DNA opposite C and then pairing with T (6,12,14,23). Apur has been reported to induce mutations in mammalian cells (2,5).…”

“…One hypothesis, based on in vivo studies with procaryotes (1,12,14,23) and in vitro studies with purified DNA polymerases and synthetic templates (27) (6). This hypothesis predicts that the concentrations of the endogenous substrates dCTP and TTP will be an important factor influencing Apur-induced mutagenesis.…”