title: The KYxxL motif in Rad17 protein is essential for the interaction with the 9-1-1 complex.
abstract: ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17-RFC and Rad9-Rad1-Hus1 (9-1-1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9-1-1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9-1-1 complex is not required for Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9-1-1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization.
authors.affiliation: Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan. Electronic address: fukumoto@faculty.chiba-u.jp.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Fukumoto
authors.affiliation: Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Ikeuchi
authors.affiliation: Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nakayama
authors.affiliation: Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan. Electronic address: nyama@faculty.chiba-u.jp.
authors.forename: Naoto
authors.initials: N
authors.lastname: Yamaguchi
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D021122:Protein Subunits; D013329:Structure-Activity Relationship
chemical_list: D018797:Cell Cycle Proteins; D021122:Protein Subunits; C113575:Rad17 protein, human; D004247:DNA
keywords: ATR; Cell cycle checkpoint; DNA damage response; Rad17; Rad9; The 9–1–1 complex