Psoralen-sensitive mutant pso9-1 of Saccharomyces cerevisiae contains a mutant allele of the DNA damage checkpoint gene MEC3

Abstract

Complementation analysis of the pso9-1 yeast mutant strain sensitive to photoactivated mono- and bifunctional psoralens, UV-light 254 nm, and nitrosoguanidine, with pso1 to pso8 mutants, confirmed that it contains a novel pso mutation. Molecular cloning via the reverse genetics complementation approach using a yeast genomic library suggested pso9-1 to be a mutant allele of the DNA damage checkpoint control gene MEC3. Non-complementation of several sensitivity phenotypes in pso9-1/mec3Δ diploids confirmed allelism. The pso9-1 mutant allele contains a −1 frameshift mutation (deletion of one A) at nucleotide position 802 (802delA), resulting in nine different amino acid residues from that point and a premature termination. This mutation affected the binding properties of Pso9-1p, abolishing its interactions with both Rad17p and Ddc1p. Further interaction assays employing mec3 constructions lacking the last 25 and 75 amino acid carboxyl termini were also not able to maintain stable interactions. Moreover, the pso9-1 mutant strain could no longer sense DNA damage since it continued in the cell cycle after 8-MOP + UVA treatment. Taken together, these observations allowed us to propose a model for checkpoint activation generated by photo-induced adducts.

Introduction

The isolation of yeast mutants specifically sensitive to photoactivated mono- and bifunctional psoralens, so-called pso mutants, was initiated by Henriques and Moustacchi (1980) in order to study possible genotoxic effects of these medically important furocoumarins that are applied in the photo-therapy of dermatological disorders like psoriasis and vitiligo [1]. Depending on their molecular structure photoactivated psoralens, upon their photo-activation with UVA (UV-light of 360 nm), may form mono- and di-adducts with thymine in DNA [2], [3], [4], [5], [6]. The di-adduct DNA lesion results from two consecutive reactions of, for example, 8-MOP with two thymine residues of opposite DNA strands and leads to DNA inter-strand cross-links (ICL). Psoralen-sensitive mutants, therefore, are putatively DNA repair-deficient. Psoralen + UVA induced ICL are very stable and may constitute one of the most toxic types of DNA damage encountered by yeast as one to two ICL per chromosome, i.e. 20 ICL contained in 16 chromosomes of a haploid Saccharomyces cerevisiae wild-type strain, are lethal [7], [8], [9]. Studies of pso mutants have contributed to our knowledge on the different interactions of photoactivated psoralen-induced DNA damage with repair functions that, when impaired or absent, may alter viability and mutagenic responses.

Phenotypic, genetical and molecular analysis of the hitherto known eight pso mutants have allowed their separation into three groups; five which either directly or indirectly play a role in the mechanisms of error-prone DNA repair, two that lack protection against reactive oxygen species (ROS) or had an altered energy metabolism, and one that represents error-free excision repair. Of the five DNA repair genes involved in induced mutagenesis two PSO loci [PSO1/REV3 and PSO8/RAD6] were allelic to already known repair genes, whereas three, PSO2/SNM1, PSO3/RNR4, and PSO4/PRP19 represent new genes involved in DNA repair and nucleic acid metabolism of S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of ICL and may have a role in non-homologous end joining [10]. The low induced mutability of pso3/rnr4 mutants indicates an important role of this subunit of ribonucleotide reductase (RNR) in regulation or function of translesion polymerase zeta in error-prone repair (reviewed in [11]). Prp19p/Pso4p influences efficiency of DNA repair via splicing of pre-mRNAs of intron-containing repair genes but also may function in the stability of the nuclear scaffold that might influence DNA repair capacity [12]. Mutations in genes PSO6 (allelic to ERG3) and PSO7 (allelic to COX11) alter the respective mutants’ sensitivities by interfering with protective mechanisms [13] or by modulating the metabolism of certain mutagens [14], respectively.

Maintaining genome integrity is the main challenge for living cells. To achieve this cells have, therefore, evolved surveillance mechanisms, the so-called checkpoints, that monitor the structure of chromosomes and coordinate DNA repair and cell-cycle progression. Such mechanisms refer to the biochemical pathways which are activated in response to internal and external aggressions (DNA damage), responsible for the induction of transcriptional programs for the inhibition of cell-cycle progression. This would allow DNA repair before completion of cell division (for reviews, see [15], [16], [17], [18]).

To extend our understanding of the genetical and biochemical basis of UVA-activated psoralen-induced DNA repair we have identified and cloned a further pso mutant from the original stock of mutagenized yeast cells [19]. In this report, we describe the phenotypic characteristics and the molecular cloning of this mutant which complemented all hitherto described pso mutant strains (pso1 to pso8). By classical and molecular genetics methods we show that the mutagen sensitivity of the yeast strain containing mutant allele pso9-1 is due to a deficiency in an important checkpoint protein, encoded by the yeast MEC3 gene.