title: The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint.
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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Vasundhara M
authors.initials: VM
authors.lastname: Navadgi-Patil
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Peter M
authors.initials: PM
authors.lastname: Burgers
mesh_terms: D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000096:biosynthesis*; D004789:Enzyme Activation; D059565:G2 Phase Cell Cycle Checkpoints / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D059566:M Phase Cell Cycle Checkpoints / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D059807:S Phase Cell Cycle Checkpoints / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; C097185:DPB11 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases