Two separate pathways regulate protein stability of ATM/ATR-related protein kinases Mec1 and Tel1 in budding yeast.

Checkpoint signaling requires two conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs): ATM and ATR. In budding yeast, Tel1 and Mec1 correspond to ATM and ATR, respectively. The Tel2-Tti1-Tti2 (TTT) complex connects to the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex for the protein stability of PIKKs; however, TTT-R2TP interaction only partially mediates ATM and ATR protein stabilization.

SGT1-HSP90 complex is required for CENP-A deposition at centromeres.

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A-containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere.

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions.

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition.

Two large phosphatidylinositol 3-kinase-related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively.

CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere.

The role of the Saccharomyces cerevisiae Cdc7-Dbf4 complex in the replication checkpoint.

The Cdc7-Dbf4 complex is a conserved serine/threonine protein kinase essential for the initiation of eukaryotic DNA replication. Although an mcm5-bob1 mutation bypasses lethality conferred by mutations in CDC7 or DBF4, the Deltacdc7 mcm5-bob1 mutant is sensitive to hydroxyurea (HU), which induces replication stress. To elucidate the reasons for HU sensitivity conferred by deletion of CDC7, we examined the role of Cdc7-Dbf4 in the replication checkpoint.