Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint.
The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcription-coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.
Giannattasio
Michele
M
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.
Lazzaro
Federico
F
Longhese
Maria Pia
MP
Plevani
Paolo
P
Muzi-Falconi
Marco
M
D048868:Adaptor Proteins, Signal Transducing; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D015825:Chromosomes, Fungal; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D005800:Genes, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009154:Mutation; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D014466:Ultraviolet Rays
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C440013:Ddc1 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; D046911:Macromolecular Substances; D010750:Phosphoproteins; C072975:RAD14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D045643:DNA Repair Enzymes