Psoralen-sensitive mutant pso9-1 of Saccharomyces cerevisiae contains a mutant allele of the DNA damage checkpoint gene MEC3.
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/mec3Delta 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.
Cardone
J M
JM
Universidade Federal do Rio Grande do Sul, Department de Biofísica/Centro de Biotecnologia, UFRGS, Av. Bento Gonçalves 9.500, Agronomia CEP, 91501 970 Porto Alegre, RS, Brasil.
Revers
L F
LF
Machado
R M
RM
Bonatto
D
D
Brendel
M
M
Henriques
J A P
JA
D000483:Alleles; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000378:metabolism; D005363:Ficusin / Q000494:pharmacology*; D017353:Gene Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005838:Genotype; D008027:Light; D008954:Models, Biological; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D009604:Nitrosoguanidines / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D003470:Culture Media; C099938:MEC3 protein, S cerevisiae; D009153:Mutagens; D009604:Nitrosoguanidines; D029701:Saccharomyces cerevisiae Proteins; D005363:Ficusin