Association of Rad9 with Double-Strand Breaks through a Mec1-Dependent Mechanism

Abstract

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1-and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1⌬ or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1⌬ mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

“…Inhibiting CDK1 counteracted degradation of the HO-cut fragment as well as additional fragments a greater distance from the DSB. Resection was not significantly affected in G2-arrested cells deleted for Rad9, Rad17 or Mec1 checkpoint proteins 12 (data not shown).…”

Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway

“…Cells carrying the rfa1-t11 mutation (55) were obtained by transforming the pKU2-t11 plasmid (obtained from K. Umezu and R. Kolodner) after linearization by PCR. Other strain constructs were described elsewhere (25,37,38,59). All the strains used in this study are isogenic to KSC1516 (37) and are listed in Table 1.…”

“…Immunoblotting was performed as described previously (37). To examine Rad53 phosphorylation after exposure to phleomycin or UV light, cells carrying YCpT-RAD53-HA were grown in yeast extract-peptone-dextrose (YEPD) and then arrested at G 1 or G 2 /M after a 2-h incubation with 6 g of ␣-factor/ml or 15 g of nocodazole/ml, respectively.…”

“…DNA degradation at DSB ends was monitored as previously described (37,38). To prepare strand-specific probes, DNA fragments were amplified from genomic DNA by PCR with oligonucleotide primers flanked by a T7 promoter sequence.…”

Activation of the Checkpoint Kinase Rad53 by the Phosphatidyl Inositol Kinase-like Kinase Mec1

“…Earlier studies suggested that Rad9 functions as an adaptor or scaffold protein for signal transmission to Rad53. After DNA damage, phosphorylated Rad9 localizes at double-strand breaks, and phosphorylated Rad9 interacts selectively with the FHA domains of Rad53 (12,27,[34][35][36][37]. Mutagenesis studies show that phosphorylation of Rad9 at PIKK consensus sites is important for checkpoint activation and interaction with Rad53 (12).…”