Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides.
Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden, and.
Paulina H
PH
Wanrooij
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Elias
E
Tannous
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
Sandeep
S
Kumar
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Vasundhara M
VM
Navadgi-Patil
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, burgers@biochem.wustl.edu.
Peter M
PM
Burgers
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D011392:Proline / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; C440013:Ddc1 protein, S cerevisiae; D020536:Enzyme Activators; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D011392:Proline; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae
DNA damage response; checkpoint control; enzyme kinetics; peptides; serine/threonine protein kinase