The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) is dispensable for DNA replication and the S Phase checkpoint but required for the G2/M checkpoint.

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 aa583 of the 764 aa 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 1-600aa domain of DPB11 is both required and sufficient for full replication function of Dpb11, but this domain is defective for activation of the principal checkpoint kinase Mec1 (human ATR) 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 G2/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 G2/M checkpoint in yeast. Together with aromatic motifs previously identified in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.
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Date: Sep. 28, 2011
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