Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication.
Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.
Ohuchi
Takashi
T
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Seki
Masayuki
M
Kugou
Kazuto
K
Tada
Shusuke
S
Ohta
Kunihiro
K
Enomoto
Takemi
T
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics*; D004271:DNA, Fungal; D005434:Flow Cytometry; D015151:Immunoblotting; D009154:Mutation / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004269:DNA, Bacterial; D004271:DNA, Fungal; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase