The Influence of Neighboring Base Pairs upon Base-Pair Substitution Mutation Rates

Abstract

The 2-aminopurine-induced transition,A T --G C, was studied at particular sites in bacteriophage T4 as a function of the nearby base-pair composition of the DNA. Changing a base pair changed the transition rate at the adjacent base pair up to 23-fold, and at the next base pair by a lesser amount. Destabilization was achieved by replacing an A -T base pair by a G C base pair.The base-pair substitution mutation rate at a particular genetic site is often thought to depend not only upon the base pair at that site, but also upon the molecular environment determined by nearby base pairs. This idea seems to have originated as an explanation of the "hot spots" observed in the pioneering studies of mutability at the r1I locus of bacteriophage T4 (1, 2). The main difficulty in interpreting the spectra of forward-and reverse-mutation rates arises from indefinite knowledge of which degenerate codon occurs at a given DNA locus, and about what amino acids are acceptable at particular positions in the polypeptide. Determinations of the amino acid compositions of many revertants of amber mutations located in the T4 head-protein gene, however, revealed differing rates of specific base-pair substitution at different positions within the cistron (3). Furthermore, T4rII ochre mutants were induced by hydroxylamine (CAA UAA) at rates that varied by as much as 20-fold at different sites (4). The ultravioletinduced reversion of ochre mutants within the yeast iso-icytochrome c gene differs at different positions: although glutamine is acceptable at two particular positions, for instance, it was induced in 11 of 11 revertants at one, and in 0 of 11 at another (5). While strongly suggestive of neighboring-base effects, results of this type are also compatible with an alternative interpretation, namely, that mutation rates vary within a cistron because of extrinsic factors such as direction of gene replication, direction of transcription, proximity to control elements, and so on. In fact, only a small number of externally determined gradients of mutability would be necessary to explain the variations in specific base-pair substitution mutation rates thus far observed. A convincing demonstration that nearby base pairs do in fact influence base-pair substitution mutation rates therefore requires that a mutation rate be experimentally altered by a nearby base-pair substitution. RATIONALEThe amber (UAG) and ochre (UAA) chain-terminating codons are mutationally interconvertible by a transition in the third position (Fig. 1). Mutation rates at the first two positions of such homologous codons can be measured, providing that the 773 phenotype corresponding to each possible genotype can be recognized with confidence. These measurements are possible using the T4rII system. First, the possible types of base-pair substitutions have been restricted by using the mutagen 2-aminopurine, which produces exclusively transitions. It is already clear that 2-aminopurine readily produces transitions, and the following evidence shows that it does not pro...

The influence of nearest neighbors on the rate and pattern of spontaneous point mutations

“…Topal et al (1980) concluded, on the basis of in vitro competition experiments with DNA polymerase, that nearest-neighbor interactions between incoming dNTPs and the terminal base of the nascent strand influence the frequency of substitution mutations. Variable frequencies of 2-aminopurine-induced transitions found by Koch (1971) suggest a dependence on sequence context. Mendelman et al (1989) found that the kinetics of nucleotide misincorporation by DNA polymerase a is dependent on nearest-neighbor base stacking.…”

Templated Mutagenesis in Bacteriophage T4 Involving Imperfect Direct or Indirect Sequence Repeats

“…The experimental modification of site-specific mutation rates by changes in neighboring bases has been studied both in vitro (Kunkel and Soni 1988;Bebenek et al 1993) and in vivo in T4, where Koch (1971) was the first to demonstrate changes in site-specific mutation rates when their neighboring bases were altered. At least in the cases of T4 (which lacks conventional DNA mismatch repair) and of DNA synthesis in vitro by various polymerases, the rate of formation and/or disposition of a mismatch during synthesis can reasonably be expected to vary as a function of its close neighbors, but the number of template and primer bases contacted by the polymerase and/or proofreading exonuclease is small, so the effects of more distant bases might be expected to extinguish quickly.…”

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria

“…Therefore, the thyP3 -based conclusion that conflicts predominantly generate promoter mutations should only be considered with these caveats in mind. All reporter systems therefore have fundamental constraints that need to be carefully considered if one is investigating mutational footprints, especially given that sequence context is well known to affect mutagenesis as well (22). So how can one accurately determine the nature of mutations caused by conflicts?…”