br Resection of DNA ends The highly
Resection of DNA ends The highly conserved MRN/MRX complex (Mre11-Rad50-Nbs1 in metazoan; Mre11-Rad50-Xrs2 in yeast) and the Ku70/Ku80 heterodimer (hereafter referred to as Ku) are the first protein complexes to be recruited at DSBs . The presence of Ku and MRN/MRX mediates the recruitment of proteins that religate the broken DNA ends by NHEJ , , . NHEJ is active only on blunt or minimally processed DNA ends, and therefore is inhibited by the nucleolytic degradation of the 5′ strands. The latter process, referred to as 5′–3′ resection, generates 3′ single-stranded DNA (ssDNA) tails at the DSB ends and commits DSB repair to HR . The Replication Protein A (RPA) complex binds to the ssDNA tails and recruits the ATR/Mec1 checkpoint kinase. Thus the decision to resect a DSB is fundamental not only to initiate DSB repair by HR, but also to activate the ATR/Mec1-mediated checkpoint response.
The ATR/Mec1 and ATM/Tel1 checkpoint kinases Generation of DNA DSBs triggers the activation of the DNA damage checkpoint signal transduction pathways, which coordinate the DNA damage response . Checkpoint signaling comprises a protein kinase cascade initiated by the two apical protein kinases, which are called ATM and ATR in mammals or Tel1 and Mec1, respectively, in S. cerevisiae (Fig. 2). In collaboration with accessory proteins, these two kinases respond to DNA damage by phosphorylating downstream effectors that coordinate 5z progression with DNA repair. ATM and ATR are members of the phosphoinositide 3-kinase-related protein kinase (PIKK) family. The consensus motif for phosphorylation by ATM/ATR is hydrophobic-X-hydrophobic-[S/T]-Q. Other members of the PIKK family include the DNA-dependent protein kinase (DNA-PK), the homolog of Caenorhabditis elegans SMG-1 (SMG1) and the mammalian target of rapamycin (mTOR). The PIKK enzymes are large proteins (270–450kDa) that have analogous structures, characterized by N-terminal HEAT repeat domains followed by relatively small kinase domains . The kinase domain is located near the C-terminus and is flanked by two regions of sequence similarity called FAT (FRAP, ATM, TRRAP) and FATC (FAT C-terminus) domains, which might interact and participate in the regulation of the kinase activity . The remaining part of each protein consists of multiple α-helical HEAT repeats , a single HEAT unit being a pair of interacting anti-parallel helices linked by a flexible “intra-unit” loop . These regions are predicted to adopt large superhelical conformations creating extended surfaces that mediate protein and DNA interactions. The presence of a common evolutionarily conserved structure among all PIKK-like proteins raises the possibility that, despite their different biological roles, all these proteins share common underlying properties. Both ATM/Tel1 and ATR/Mec1 are activated by DNA damage, but their DNA damage specificities are distinct. ATM/Tel1 is activated by DSBs, whereas ATR/Mec1 responds to a broad spectrum of DNA lesions that induces the generation of ssDNA . DNA damage-activated ATR/Mec1 and/or ATM/Tel1 promote the activation of the downstream effector kinases Rad53 and Chk1 (vertebrate CHK2 and CHK1, respectively) . In S. cerevisiae, Mec1 activates both Rad53 and Chk1, while human ATM and ATR primarily activate CHK2 and CHK1, respectively. Activation of the effector kinases requires mediator proteins, among which are the BRCT-domain-containing protein Rad9 and its metazoan ortholog 53BP1 (Fig. 2). In particular, Rad9 is phosphorylated in a Mec1- and/or Tel1-dependent manner upon DNA damage, and these phosphorylation events create a binding site for Rad53, which then undergoes in-trans autophosphorylation events required for its full activation as a kinase , , . Mec1-dependent phosphorylation of Rad53 allows further autoactivation , . Moreover, Rad9 oligomerization is required to maintain checkpoint signaling through a feedback loop involving Rad53-dependent phosphorylation of the Rad9 BRCT domain . Fully activated Rad53 is then released from the hyperphosphorylated Rad9 complex . Vertebrate CHK2 is also known to dimerize and to undergo ATM-dependent autophosphorylation in-trans, but the role for the DNA damage mediators in this activation remains to be investigated.