Cooperative control of Crb2 by ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast.
The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as gamma-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that gamma-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in gamma-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.
Nakamura
Toru M
TM
Department of Molecular Biology, MB3, The Scripps Research Institute, La Jolla, CA 92037, USA.
Moser
Bettina A
BA
Du
Li-Lin
LL
Russell
Paul
P
D019943:Amino Acid Substitution / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D025521:Tumor Suppressor Proteins; D013912:Threonine; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C469447:cdc2 protein, S pombe