Regulation of the Rad53 protein kinase in signal amplification by oligomer assembly and disassembly.
Rad53, the ortholog of mammalian Chk2, is a major DNA damage checkpoint effector kinase in Saccharomyces cerevisiae. Despite extensive studies on the genetic requirements for Rad53 activation and its linkage downstream to checkpoint responses, the mechanism of Rad53 activation is not completely understood. Rad53-dependent signal amplification is thought to be a primary force that accelerates checkpoint signal transduction processes in response to DNA damage. Rad53 forms oligomers upon DNA damage in vivo. It is not clear how oligomer formation affects Rad53 activation and what is the mechanism of Rad53 oligomerization. Here, we monitor Rad53 oligomer assembly and disassembly in vitro. These processes are ATP-dependent and are regulated through phosphorylation. Mutations in FHA or SCD domains of RAD53 compromise intermolecular autophosphorylation activity and these domains are indispensable for Rad53 oligomerization. The mediator Rad9 is not necessary for Rad53 oligomerization. Rad53 kinase activity is required for disassembly of Rad53 oligomers in vivo after DNA damage. Moreover, induced oligomerization of Rad53 efficiently activates Rad53 in the absence of Mec1 in vivo. The results support the conclusions that Rad53/Chk2 homo-oligomerization is an evolutionarily conserved mechanism that drives Rad53/Chk2 activation and promotes signal amplification in DNA damage responses.
Jia-Lin Ma
Nianhan
N
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Stern
David F
DF
D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004789:Enzyme Activation / Q000502:physiology*; D047468:Immunoprecipitation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae