Copyright 1998 by the Genetics Society of America DNA Sequence Analysis of Spontaneous Mutagenesis in Saccharomyces cerevisiae Bernard A. Kunz, Karthikeyan Ramachandran and Edward J. Vonarx School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria, 3217, Australia ABSTRACT To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively. Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis. In this article, we survey and discuss the mutational speci®cities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae. The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis. PONTANEOUS mutations play a fundamental role in the production of single and multiple base pair alter- S in evolution and have been implicated in aging, ations (Ripley and Glickman 1983; Kunkel 1990). In- carcinogenesis, and human genetic disease (Harmon sertions of transposable elements generally constitute 1981; Kirkwood 1989; Cooper and Krawczak 1990; larger sequence changes in DNA. Although transposon Arber 1991; Drake 1991a; Loeb 1991, 1994; Win- movement can be a relatively infrequent event, transpo- tersberger 1991; Caskey et al. 1992; Strauss 1992). sition rates may be increased by the presence of DNA They are thought to originate as a consequence of intra- damage, including that which occurs spontaneously cellular events, including the formation of DNA lesions, (Bradshaw and McEntee 1989; Kunz et al. 1990, the occurrence of DNA synthesis errors during replica- 1994a). tion, repair and recombination, and the movement of Two general strategies have been used in attempts transposable elements (Sargentini and Smith 1985; to better understand the mechanisms responsible for Smith and Sargentini 1985; Ramel 1989; Loeb and spontaneous mutagenesis. The ®rst is to characterize Cheng 1990; Drake 1991b; Kunkel 1992; Smith 1992; strains that have enhanced spontaneous mutation rates. Amariglio and Rechavi 1993; Ames et al. 1993; Lin- The rationale for this route is that mutator phenotypes dahl 1993). are expected to result from defects in genes whose prod- DNA lesions can arise naturally through intracellular ucts act to minimize genetic instability. Indeed, as ex- metabolism or the intrinsic instability of DNA. For exam- pected, such studies have revealed that spontaneous ple, there is evidence consistent with spontaneous alkyl- mutations can arise through failure of DNA repair or ation, deamination, and loss of DNA bases, as well as processes that maintain the accuracy of DNA replica- Loeb their modi®cation by reactive oxygen species ( and tion (Haynes and Kunz 1981; Sargentini and Smith Preston Ames Lindahl Fried- 1986; et al. 1993; 1993; 1985; Kramer et al. 1989a; Rebeck and Samson 1991; berg et al. 1995). Because a substantial fraction of en- Michaels and Miller 1992; Smith 1992; Xiao and dogenous DNA damage may involve the alteration or Samson 1993; Kunz et al. 1994a and references therein). loss of nucleotide bases, many spontaneous mutations The second approach is to isolate antimutator mutants may be generated during replication past miscoding or with the aim to identify genes whose functions are re- noninstructional lesions. The accuracy of DNA synthesis quired for spontaneous mutagenesis. A number of these on undamaged templates is attributed in large measure mutants have been recovered, mainly in prokaryotic to DNA polymerase (pol)-mediated nucleotide selection systems, and where characterized, they have been found and proofreading of replication errors, as well as to to have alterations primarily in genes that encode DNA postreplicative mismatch correction of errors that es- polymerases (Sargentini and Smith 1985; Morrison cape proofreading (Loeb and Cheng 1990; Kunkel et al. 1989; Smith 1992; Drake 1993 and references 1992; Goodman et al. 1993; Modrich 1994). The topog- therein; Fijalkowska et al. 1993). raphy of the DNA molecule itself also is an important Early studies of spontaneous mutagenesis in mutator factor, however, and roles have been proposed for DNA and antimutator strains relied on genetic analysis of secondary structures and misaligned template primers reversion, suppression, or forward mutation (for re- views, see Lawrence 1982; Sargentini and Smith 1985; Friedberg et al. 1995). Because of technological Corresponding author: B. A. Kunz, School of Biological and Chemi- cal Sciences, Deakin University, Geelong, Victoria, 3217 Australia. restrictions, the systems used did not identify the DNA E-mail: [email protected] sequence alterations involved, or they were unable to Genetics 148: 1491±1505 (April, 1998) 1492 B. A. Kunz, K. Ramachandran and E. J. Vonarx detect all types of mutation that occurred. These limita- large number of samples. In this case, however, geno- tions were overcome by applying DNA sequencing tech- typic and phenotypic reversion to wild type are assumed niques to examine forward mutations in prokaryotic to involve the same sequence change, but this may not and then eukaryotic organisms (Friedberg et al. 1995). necessarily be so. For example, intragenic suppression The resulting data on the types of DNA sequence may be indistinguishable phenotypically from locus re- changes that occur, the rates at which they arise, their version. Reversion systems will notbe considered further site speci®city, and how these parameters are in¯uenced in this review. by mutator and antimutator alleles have yielded insights Mutational spectra derived from different assay sys- into processes that act to maintain genetic stability and tems should be compared with caution because of the ensure accurate transmission of the hereditary material. inherent characteristics of the systems. Ideally an assay In this article, we review the modulation of spontane- system should involve a target gene large enough to ous mutagenesis in mutator and antimutator strains of allow all possible types of mutation to occur and be the budding yeast Saccharomyces cerevisiae, as revealed recovered. While a larger target gene should be more by DNA sequence analysis. First, we discuss brie¯y the representative of the genome, practical dif®culties in selected aspects of systems used in the analysis of muta- sequencing could negate this advantage. Multiple se- genesis. Next, sequence changes arising spontaneously quencing sessions may be required to identify mutations in a wild-type background are considered to establish in a large target. Even if a mutation is identi®ed near the types of mutations that do occur and provide a basis the sequencing primer, the entire target has to be se- for comparison. We then survey the effects of eliminat- quenced to verify that there are no additional alter- ing genes whose inactivation leads to a mutator or anti- ations. Another desirable feature is a wide variety of mutator phenotype on the magnitude and speci®city of sequence contexts, which helps maximize detection of spontaneous mutagenesis. Finally, we focus on a select sequence-speci®c effects. The proportion of intronic number of ®ndings with implications for the mecha- sequences in the assay system, however, should be low nisms of spontaneous mutagenesis in eukaryotic cells. because many intronic mutations may not produce phe- Assay systems: In vivo mutagenesis assay systems can notypic changes. If nondetectable mutations are not be based on the characterization of forward or reverse randomly distributed with respect to class or location mutations on a target gene (assay gene). Sequence anal- throughout the target gene, then mutational speci®city yses using forward systems have been reported for the will be biased and the mutational data will not be repre- CAN1, LYS2, SUP4-o, and URA3 genes. Commonly used sentative of the true spectrum. Thus, if possible, the reversion systems are based on ade2, his7, hom3,orlys2 ability of the system to detect all possible substitutions alleles. Mutant strains are usually constructed by delet- at all positions within the target should be characterized. ing part or all of the gene, disrupting the gene sequence If this requirement is not met, the interpretation of by inserting additional DNA into the gene, or by intro- the spectral data should acknowledge this limitation. ducing point mutations into the gene. Deletion of a Irrespective of the size or sequence content of the assay signi®cant portion or all of the gene is the preferable target, a statistically signi®cant number of mutants must method because this usually results in complete loss of be examined to construct a meaningful spectrum. Small the gene's function. In contrast, disruptions and point data sets increase the probability that spectral features mutations may result in partial retention or alteration of are caused by chance. gene function, and different point mutations especially Spontaneous DNA sequence alterations: To date, mu- may produce different phenotypic effects. Assay genes tations that occur spontaneously in wild-type cells have can be located on a chromosome or a centromeric plas- been characterized using the yeast centromere plasmid- mid. This type of plasmid mimics chromosome behavior borne SUP4-o