The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.Checkpoints arrest cell cycle progression in the presence of DNA damage to facilitate DNA repair and prevent genetic instability (1-3). There is now a considerable amount of information available that characterizes signal transmission mechanisms and downstream targets in lower and higher eukaryotic cells. However, the precise roles of the initial damage-sensing proteins and their relevant DNA or chromatin substrates have remained largely elusive. Characterization of candidate proteins supports the notion that the underlying mechanisms are conserved throughout eukaryotic evolution (4 -7). Consequently, the yeast Saccharomyces cerevisiae will continue to play an important role as a relevant model system.A damage sensor function has been suggested for the PCNA 1 -like complex composed of the budding yeast proteins Rad17p, Ddc1p, and Mec3p, also termed 9-1-1 complex (following the nomenclature adapted for human cells and Schizosaccharomyces pombe) (8 -11). Although direct evidence is still missing, it is likely that this ring-shaped complex (12) encircles DNA and is loaded onto DNA by a replication factor C-related complex in which the largest replication factor C subunit is replaced by the checkpoint protein Rad24p (13). The phosphoinositol kinase-related protein Mec1p (an ortholog of the human ATR protein) has been identified as an independent damage-sensor candidate (9, 11). Mec1p forms a complex with Lcd1p, a protein with an affinity for DNA double-stranded breaks, and thus Mec1p kinase activity can be targeted to sites of DNA damage (14). Mec1p and its downstream target Rad53p are involved in virtually all DNA damage and replicationrelated checkpoints.Other potential sensor proteins responding to doublestranded breaks are represented by the Rad50p⅐Xrs2p⅐Mre11p complex. This complex plays a well established role in nonhomologous endjoining and homologous recombination, whereas a function in checkpoint arrest following treatment with strand break-inducing agents has only recently been uncovered (15, 16).Although such putative damage recognition complexes appear to be formed independently from each other, their combined action is frequently necessary for the generation of a downstream signal. A key playe...
