We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of g3-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G-C --TA transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the IacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in ANT -C-G transversions.Mutants with higher than normal rates of spontaneous mutation have facilitated our understanding of mutational pathways. Some of the "mutator" strains have characterized defects in postreplication mismatch repair (1, 2), in specific glycosylases (3), or in the editing function provided by the e subunit of DNA polymerase III (4). Characterization of additional mutators may reveal pathways of mutagenesis and repair. Toward this end, we have used a highly sensitive screening method to detect mutator strains that revert defined mutations in lacZ by a limited number of base substitutions. Here we report the characterization of a mutator locus, mutY, which results in the specific generation of G-C

We used strains carrying specific lacZ alleles to identify a new mutator locus in Escherichia coli which generates only G. C -*T T A transversions among base substitutions. The locus, mutM, mapped near the cysE locus, which is at 81 min on the genetic map.The pathways that lead to spontaneous base substitution mutations have not been completely defined. Although we know that replication errors and spontaneous lesions are potential sources of mutation, it is not certain which is the principal cause of each of the different spontaneous base substitutions that are observed. In particular, the pathways that lead to transversion mutations are still somewhat obscure. It is not clear how transversions occur as replication errors (see, for instance, reference 18) and how many repair systems exist which can efficiently correct these errors. The methyl-directed mismatch repair system encoded by the Escherichia coli mutHLS and uvrD genes (11, 14) can correct mispairings that lead to both transitions and most transversions (4)(5)(6) 17). Yet, strains lacking this system display more greatly enhanced levels of transitions than transversions (2, 8, 15,16). This might simply be a reflection of the levels of each type of mispair generated during replication, or it might signal the existence of additional repair systems. Is there a system that specifically corrects mispairings that lead to transversions?We have recently described a mutator locus in E. coli, mutY, which results in G C -* T. A transversions (13). and Lu and Change (10) have described a DNA mismatch correction activity in vitro that specifically repairs G A mispairs, resulting in G C base pairs, and which is independent of the mutHLS-dependent pathway. Strains which carry mutY lose the mutHLS-independent repair activity specific for G A mispairs (K. G. Au, P. Modrich, M. Cabrera, and J. H. Miller, unpublished results), demonstrating that this activity represents a repair system that functions in vivo as a mechanism for avoiding G C --T. A transversions. These results also demonstrate the value of studying mutators as a way of elucidating mutagenic pathways.Detection of new mutator strains. We looked for additional mutator strains that might have defects in the same or different pathways responsible for transversions. Here we describe a second locus, mutM, involved in the avoidance of G. C -* T. A transversions. We detected this locus by mutagenizing a strain, CC104 (13), that carries a specific mutation in lacZ and then screening for colonies with an increased reversion rate to Lac'. The mutation carried in strain CC104, which changes the active-site glutamic acid residue at position 461 in beta-galactosidase to alanine, resulting in a defective enzyme is shown in Fig. 1. Only the * Corresponding author.G-C --T-A change can restore the glutamic acid residue, which is essential for sufficient beta-galactosidase activity to allow growth on lactose (C. G. Cupples and J. H. Miller, Genetics, in press). Revertants to Lac' are detected by papillation on indicator media...

A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of

We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.