Abstract
Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.
Highlights
Specific activators turn on Mec1/ATR kinase during DNA damage or replication stress, mediating cell cycle arrest
We have identified the motifs in Dpb11 that are important for the activation of Mec1 kinase activity, and we have generated domain mutants that separate the replication function of Dpb11 from its checkpoint activation function
We show that mutants of Dpb11 that are proficient for DNA replication but deficient for Mec1 activation have no effect on the replication checkpoint
Summary
Specific activators turn on Mec1/ATR kinase during DNA damage or replication stress, mediating cell cycle arrest. This model could explain why other replication initiation mutants with defects that may cause a decrease in the number of replication forks during S phase would be defective in the replication checkpoint after nucleotide depletion, e.g. by hydroxyurea These considerations are pertinent to the case of DPB11 because Dpb is both actively involved in replisome assembly and in checkpoint activation. The dpb mutant is sensitive to hydroxyurea treatment and shows decreased checkpoint activation in response to hydroxyurea, as measured by Rad phosphorylation [17] Whether this checkpoint defect is caused directly by the inability of Dpb to activate Mec in S phase, or is an indirect effect caused by defective replisome assembly in dpb remains to be established. We conclude from our studies that the replication checkpoint defects observed previously with the dpb allele are an indirect effect of defects in replisome assembly that in itself leads to an inefficient replication checkpoint response
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