DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells.
The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.
Giannattasio
Michele
M
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Lazzaro
Federico
F
Siede
Wolfram
W
Nunes
Elia
E
Plevani
Paolo
P
Muzi-Falconi
Marco
M
D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics* / Q000502:physiology; D045643:DNA Repair Enzymes; D004271:DNA, Fungal / Q000378:metabolism / Q000528:radiation effects; D004268:DNA-Binding Proteins; D016193:G1 Phase / Q000235:genetics / Q000502:physiology; D016195:G2 Phase / Q000235:genetics / Q000502:physiology; D007399:Interphase / Q000235:genetics / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion / Q000235:genetics; D014466:Ultraviolet Rays; D014867:Water / Q000494:pharmacology
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C072975:RAD14 protein, S cerevisiae; C501395:RAD17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D045643:DNA Repair Enzymes