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1.
Genet Mol Res ; 7(1): 127-32, 2008 Feb 12.
Article in English | MEDLINE | ID: mdl-18273828

ABSTRACT

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32Delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , DNA, Fungal/genetics , DNA-Directed DNA Polymerase/classification , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae/genetics
2.
Genet. mol. res. (Online) ; 7(1): 127-132, Jan. 2008. ilus, tab
Article in English | LILACS | ID: lil-553779

ABSTRACT

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.


Subject(s)
Cell Cycle Proteins , DNA-Directed DNA Polymerase/metabolism , DNA Repair , Phosphoproteins/metabolism , Nuclear Proteins/metabolism , DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Proliferating Cell Nuclear Antigen/metabolism , DNA-Directed DNA Polymerase/classification , DNA, Fungal , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics
3.
DNA Repair (Amst) ; 5(2): 163-71, 2006 Feb 03.
Article in English | MEDLINE | ID: mdl-16202664

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/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.


Subject(s)
Cell Cycle Proteins/genetics , DNA Damage , Ficusin/pharmacology , Mutation , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Alleles , Cell Cycle , Cloning, Molecular , Culture Media/pharmacology , DNA Repair , Dose-Response Relationship, Radiation , Escherichia coli/metabolism , Gene Deletion , Genes, Fungal , Genetic Complementation Test , Genotype , Light , Models, Biological , Mutagens/pharmacology , Nitrosoguanidines/chemistry , Phenotype , Plasmids/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolism , Time Factors , Two-Hybrid System Techniques
4.
Nucleic Acids Res ; 30(22): 4993-5003, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12434004

ABSTRACT

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.


Subject(s)
Fungal Proteins/genetics , Fungal Proteins/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Alleles , Amino Acid Sequence , Amino Acid Substitution , DNA Damage , DNA Repair , Fungal Proteins/chemistry , Genes, Fungal , Molecular Sequence Data , Mutation , Phenotype , RNA Precursors/metabolism , RNA Splicing , RNA Splicing Factors , RNA, Fungal/metabolism , Saccharomyces cerevisiae/radiation effects , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Spliceosomes , Spores, Bacterial , Temperature , Two-Hybrid System Techniques , Ultraviolet Rays
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