Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.
We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle. Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions. In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1). Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase. Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates. Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.
Neecke
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Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, 20133 Milano, Italy.
Lucchini
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Longhese
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MP
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase; D017353:Gene Deletion; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C072975:RAD14 protein, S cerevisiae; C088385:RAD24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D045643:DNA Repair Enzymes