Erich Heidenreich

Non‐homologous end joining as an important mutagenic process in cell cycle‐arrested cells

Resting cells experience mutations without apparent external mutagenic influences. Such DNA replication‐independent mutations are suspected to be a consequence of processing of spontaneous DNA lesions. Using experimental systems based on reversions of frameshift alleles in Saccharomyces cerevisiae, we evaluated the impact of defects in DNA double‐strand break (DSB) repair on the frequency of replication‐independent mutations. The deletion of the genes coding for Ku70 or DNA ligase IV, which are both obligatory constituents of the non‐homologous end joining (NHEJ) pathway, each resulted in a 50% reduction of replication‐independent mutation frequency in haploid cells. Sequencing indicated that typical NHEJ‐dependent reversion events are small deletions within mononucleotide repeats, with a remarkable resemblance to DNA polymerase slippage errors. Experiments with diploid and RAD52‐ or RAD54‐deficient strains confirmed that among DSB repair pathways only NHEJ accounts for a considerable fraction of replication‐independent frameshift mutations in haploid and diploid NHEJ non‐repressed cells. Thus our results provide evidence that G0 cells with unrepressed NHEJ capacity pay for a large‐scale chromosomal stability with an increased frequency of small‐scale mutations, a finding of potential relevance for carcinogenesis.

Adaptive Mutation in Saccharomyces cerevisiae

ABSTRACT Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.

The nuclear actin-related protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes

A mutational analysis of the essential nuclear actin‐related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide‐sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their ‘suppressor of Ty’ (SPT) phenotype, ob‐served with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome‐wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress‐responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutants enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress‐induced genes under non‐stress conditions.

Replication-dependent and selection-induced mutations in respiration-competent and respiration-deficient strains of Saccharomyces cerevisiae

Abstract Adaptive or selection-induced mutations are defined as mutations that occur in non-dividing cells as a response to prolonged non-lethal selective pressure such as starvation for an essential amino acid. In the absence of DNA replication, the processing of endogenous DNA lesions by repair enzymes probably acts as a source of mutations. We are studying selection-induced reversions of frameshift alleles in the eukaryote Saccharomyces cerevisiae. Here we show that respiration-deficient strains, totally devoid of mitochondrial DNA, yield selection-induced mutants at slightly elevated frequencies compared to isonucleic respiration-competent strains. Therefore factors of mitochondrial origin such as reactive oxygen species or hypothetical recombinogenic DNA fragments are unlikely to be mediators of selection-induced nuclear frameshift mutation in yeast. Furthermore we compared sequence spectra of reversions of the +1 hom3-10 frameshift allele and found a strong preference for −1 deletions in mononucleotide repeats in selection-induced and replication-dependent revertants, indicating slippage errors during DNA repair synthesis as well as during DNA replication. Remarkably, a higher degree of variation in the site of the reverting frameshift and accompanying base substitutions was found among selection-induced revertants.

Adaptive reversions of a frameshift mutation in arrested Saccharomyces cerevisiae cells by simple deletions in mononucleotide repeats.

Adaptive mutations are characterised as the outcome of an as yet unknown mechanism, which allows a few individuals of a cell population to overcome a starvation-induced cell cycle arrest and to proliferate. A release from such a non-lethal growth limitation is accomplished by mutations generated without DNA replication. Originally adaptive mutations were described in Escherichia coli, but more recently also in a simple eukaryote, the budding yeast Saccharomyces cerevisiae. We are studying the adaptive reversion of a frameshift allele which occurs when an auxotrophic yeast strain is starved for the amino acid essential for its proliferation. In this communication, we report on the DNA sequences from the locus concerned. Comparison between sequences from revertant clones which arose several days after growth arrest by starvation and those from revertants produced during proliferation shows significantly different mutation spectra: for replication-dependent revertants nucleotide gains and losses in a variety of sequence contexts are reasonably balanced, whereas for the replication-independent, i.e. adaptive, revertants mainly simple deletions in mononucleotide repeats were observed. These mutations resemble those known to originate from DNA polymerase slippage errors which were miscorrected or had escaped correction by the mismatch repair machinery. Our data present strong evidence for differences in the mechanistic origins of adaptive versus DNA replication-dependent mutations in a eukaryote. Most probably, mutations in non-replicating cells contribute to evolution, and if conserved in mammals, to human carcinogenesis.

The relevance of oxidative stress and cytotoxic DNA lesions for spontaneous mutagenesis in non-replicating yeast cells

Mutations arising during times of cell cycle-arrest may considerably contribute to aging and cancerogenesis. Endogenous oxidative stress could be one of the major triggers for these mutations. We used Saccharomyces cerevisiae cells, arrested by starvation for the essential amino acid lysine, to study the occurrence of reactive oxygen species (ROS), abasic (AP) sites and double strand breaks (DSBs). Furthermore, we analyzed the mutation frequencies in resting wild type cells and in cells deficient for Apn1 (with an impaired base excision repair) or Dnl4 (with an inactivated non-homologous end joining (NHEJ) DSB repair pathway) by monitoring reversions of an auxotrophy-causing frameshift in the LYS2 gene. By fluorescence methods, we observed a distinct increase of ROS-affected cells in the course of starvation-induced cell cycle-arrest. In addition, we could reveal that AP sites and DSBs accumulated under these conditions. The frequency of spontaneous frameshift mutations in wild type cells was decreased to 50% upon addition of 6mM N-acetyl cysteine. However, this radical scavenger had no effect in Dnl4-deficient cells. Our results support the hypothesis that (via an active NHEJ DSB repair pathway) the incidence of spontaneous frameshift mutations in a cell cycle-arrested state is considerably governed by oxidative stress.

Starvation for a specific amino acid induces high frequencies of rho- mutants in Saccharomyces cerevisiae.

Abstract Auxotrophic yeast cells were starved on solid media for their respective essential amino acid in the course of “adaptive mutation” experiments. Thereby, high proportions of mitochondrially respiratory deficient (rho−) mutants accumulated among the cells stressed on selective plates. Using a strain with a plus-four frameshift mutation in a chromosomal gene involved in lysine biosynthesis, we observed that many of the revertant colonies which arose late under the selective pressure were composed of mixtures of rho+ and rho− cells, indicating that they originated from founder cells containing intact as well as defective mitochondrial genomes. We show that in spite of the slower growth of rho− cells the late-appearing colonies cannot be interpreted as descending from rho− revertants present before selective plating.

A mutation-promotive role of nucleotide excision repair in cell cycle-arrested cell populations following UV irradiation

Growing attention is paid to the concept that mutations arising in stationary, non-proliferating cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of cells to UV irradiation during prolonged cell cycle arrest resulted in an increased incidence of mutations. In the present work, we determined the influence of defects in the nucleotide excision repair (NER) pathway on the incidence of UV-induced adaptive mutations in stationary cells. The mutation frequency was decreased in Rad16-deficient cells and further decreased in Rad16/Rad26 double-deficient cells. A knockout of the RAD14 gene, the ortholog of the human XPA gene, even resulted in a nearly complete abolishment of UV-induced mutagenesis in cell cycle-arrested cells. Thus, the NER pathway, responsible for a normally accurate repair of UV-induced DNA damage, paradoxically is required for the generation and/or fixation of UV-induced frameshift mutations specifically in non-replicating cells.

Glucose starvation as a selective tool for the study of adaptive mutations in Saccharomyces cerevisiae

Mutations not only arise in proliferating cells but also in resting - thus non-replicating - cells. Such stationary-phase mutations may occasionally enable an escape from growth repression and e.g. contribute to cancerogenesis or development of drug resistance. The most widely used condition for the study of such adaptive mutations in the eukaryotic model organism Saccharomyces cerevisiae is the starvation for a single amino acid. To overcome some limitations of this experimental setup we developed a new adaptive mutation assay that allows a screening for mutagenic processes during a more regular cell cycle arrest induced by the lack of a fermentable carbon source. We blocked one essential step of gluconeogenesis by inactivation of the FBP1 gene. This drives the cells into a cell cycle arrest when glucose is not available in the medium although a non-fermentable carbon source is present. As another component of the new mutation assay, we established a custom-designed test allele that contains a microsatellite sequence as a target for mutations. We demonstrated the feasibility and validity of this novel experimental setup by the observation and characterization of adaptive mutants.