DNA repair: how to PIKK a partner

In eukaryotes, members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family co-ordinate the cellular response to DNA damage. But how do these important kinases detect DNA damage and relay this information to the DNA repair and checkpoint machinery?     

Emerging common themes in regulation of PIKKs and PI3Ks

Phosphatidylinositol‐3 kinase‐related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C‐terminal regions, referred to as PIKK‐regulatory domain (PRD) and FRAP‐ATM‐TRRAP‐C‐terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol‐3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C‐terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop.     

Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities

ATR is an essential protein that functions as a damage sensor and a proximal kinase in the DNA damage checkpoint response in mammalian cells. It is a member of the phosphoinositide 3-kinase-like kinase (PIKK) family, which includes ATM, ATR, and DNA-dependent protein kinase. Recently, it was found that ATM is an oligomeric protein that is converted to an active monomeric form by phosphorylation in trans upon DNA damage, and this raised the possibility that other members of the PIKK family may be regulated in a similar manner. Here we show that ATR is a monomeric protein associated with a smaller protein called ATRIP with moderate affinity. The ATR protein by itself or in the form of the ATR-ATRIP heterodimer binds to naked or replication protein A (RPA)-covered DNAs with comparable affinities. However, the phosphorylation of RPA by ATR is dependent on single-stranded DNA and is stimulated by ATRIP. These findings suggest that the regulation and mechanism of action of ATR are fundamentally different from those of the other PIKK proteins.     

Activation of ATR and related PIKKs

The DNA damage response kinase ATR is an essential regulator of genome integrity. TopBP1 functions as a general activator of ATR. We have recently shown that TopBP1 activates ATR through its regulatory subunit ATRIP and a PIKK regulatory domain (PRD) located adjacent to its kinase domain. This mechanism of ATR activation is conserved in the S. cerevisiae ortholog Mec1. ATR is a member of the PIKK family of protein kinases that includes ATM, DNA-PKcs, mTOR, and SMG1. The PRD regulates the kinase activity of other PIKKs and may serve as a site of interaction between these kinase and their respective activators. Activation of ATR by TopBP1 is maximal at low substrate concentrations and declines exponentially as substrate concentration increases. These data are consistent with a model in which TopBP1 acts to alter the conformation of ATR-ATRIP to increase the ability of ATR to bind substrates. A further understanding of the mechanism of ATR activation will likely provide insights into the regulation of related PIK kinases.     

Mec1 function in the DNA damage response does not require its interaction with Tel2

The essential, conserved Tel2 protein plays a role in the response to DNA damage and replication stress in a wide range of eukaryotes. Tel2 interacts physically with multiple members of the PI3-kinase related protein kinase (PIKK) family in mammalian cells and fission yeast. In mammalian cells, loss of Tel2 leads to destabilization of PIKKs. Our previous work in the yeast Saccharomyces cerevisiae showed that Tel2 interacts with the PIKK Tel1 (yeast ATM kinase), and that this interaction is abrogated by the only known non-lethal TEL2 mutation in S. cerevisiae, tel2-1. We showed that this mutation specifically disrupts the function of Tel1 and not the function of the closely related protein Mec1 (yeast ATR kinase) in DNA damage responses. Here we show that Tel2 and Mec1 interact in S. cerevisiae, and that surprisingly, this physical interaction is also disrupted by the tel2-1 mutation. Although the tel2-1 mutation leads to moderately lower Mec1 levels, the ability of Mec1 to localize to a site of DNA damage and to function in DNA damage signaling remains intact. These results suggest that the model of Tel2 as solely a global regulator of PIKK stability is insufficient. Rather, Tel2 can specifically and differentially regulate the function of individual PIKKs.     

Signal transduction: how rad53 kinase is activated

Recent studies have elucidated the activation mechanism of the Rad53 checkpoint kinase and the role of Rad9-like adaptor proteins in mediating signal transduction from PIKK sensor kinases that detect DNA damage to the effector kinases that play a part in mending that damage.     

ATM、ATR和DNA损伤介导的细胞周期阻滞

ATM和ATR属于PIKK家族,是DNA损伤检查点的主要成员。它们被不同类型的DNA损伤所激活,通过磷酸化相应的下游蛋白Chk1和Chk2等,调节细胞周期各个检查点,引起细胞周期阻滞,使DNA损伤得以修复。ATM和ATR在维持基因组的稳定性中起到至关重要的作用。本文着重综述有关ATM和ATR在DNA损伤介导的细胞周期阻滞中发挥的作用以及相互关系的最新研究进展。     

Identification and characterization of FUS/TLS as a new target of ATM

ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase), important regulators of genome stability, belong to the PIKK (phosphoinositide 3-kinase-like kinase) family of protein kinases. In the present study, DNA-affinity chromatography was used to identify DNA-binding proteins phosphorylated by these kinases. This resulted in the identification of FUS (fused in sarcoma)/TLS (translocated in liposarcoma) as an in vitro target of the PIKKs. FUS is a member of the Ewing's sarcoma family of proteins that appears to play a role in regulating genome stability, since mice lacking FUS show chromosomal instability and defects in meiosis. The residues in FUS that are phosphorylated in vitro and in vivo were identified, and phospho-specific antibodies were generated to demonstrate that FUS becomes phosphorylated at Ser(42) in vivo, primarily in response to agents that cause DSBs (double-strand breaks). DSB-induced FUS phosphorylation in vivo at Ser(42) requires ATM and not DNA-PK. Although Ser(42) is retained in the oncogenic FUS-CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein 10] fusion generated by a t(12;16)(q13;p11) chromosomal translocation, Ser(42) in FUS-CHOP is not phosphorylated after DNA damage. These results identify FUS as a new target of the ATM-signalling pathway and strengthen the notion that FUS regulates genome stability.     

Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay

Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.     

Initiating cellular stress responses

The phosphoinositide 3-kinase related kinases (PIKKs) mediate responses to diverse stresses, including DNA double-strand breaks (DSBs), abnormal replication fork progression, the recognition of premature termination codons in mRNAs, and inadequate nutrient availability. Rigorous control of these kinases limits cellular damage and promotes cell viability in the presence of stress. Control mechanisms include the localization of PIKKs into multiprotein complexes at the sites of damage and mediation of protein-protein contacts such that substrates are allowed access to the PIKK catalytic domains.     

Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)

DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have now generated an approximately 13 A three-dimensional map of DNA-PKcs, revealing the overall architecture and topology of the 4128 residue polypeptide chain and allowing location of domains. The highly conserved C-terminal PIKK catalytic domain forms a central structure from which FAT and FATC domains protrude. Conformational changes observed in these domains on DNA binding suggest that they transduce DNA-induced conformational changes to the catalytic core and regulate kinase activity. The N-terminal segments form long curved tubular-shaped domains based on helical repeats to create interacting surfaces required for macromolecular assembly. Comparison of DNA-PKcs with another PIKK DNA repair factor, ATM, defines a common architecture for this important protein family.     

The FATC domains of PIKK proteins are functionally equivalent and participate in the Tip60-dependent activation of DNA-PKcs and ATM

Members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including the ATM, DNA-PKcs, Atr, and Trrap proteins, function in signal transduction pathways that activate the DNA damage response. PIKK proteins contain a conserved C-terminal FAT/kinase domain/FATC domain structure. The FATC domain of ATM mediates the interaction between ATM and Tip60, a histone acetyltransferase that regulates activation of ATM. Here, we examined whether the FATC domains of DNA-PKcs, Atr, and Trrap were also able to interact with Tip60. Deletion of the FATC domain of ATM blocked the interaction between ATM and Tip60 and suppressed the activation of ATM kinase activity by DNA damage. Replacement of the FATC domain of ATM with the FATC domains of DNA-PKcs, Atr, or Trrap restored the activation of ATM and its association with Tip60. These results indicate that the FATC domains of DNA-PKcs, Atr, Trrap, and ATM are functionally equivalent. Immunoprecipitation experiments demonstrated that Tip60 is constitutively associated with DNA-PKcs and that the histone acetyltransferase activity associated with DNA-PKcs is up-regulated by DNA damage. When Tip60 expression was suppressed by small interfering RNA, the activation of DNA-PKcs (measured by autophosphorylation of DNA-PKcs at serine 2056 and threonine 2609) was inhibited, demonstrating a key role for Tip60 in the activation of DNA-PKcs by DNA damage. The conserved FATC domain of PIKK proteins may therefore function as a binding domain for the Tip60 histone acetyltransferase. Further, the ability of Tip60 to regulate the activation of both ATM and DNA-PKcs in response to DNA damage demonstrates that Tip60 is a key component of the DNA damage-signaling network.     

A novel Tel1/ATM N-terminal motif, TAN, is essential for telomere length maintenance and a DNA damage response

Tel1/ATM, a conserved phosphatidylinositol 3-kinase-related kinase (PIKK), acts in the response to DNA damage and regulates telomere maintenance. PIKK family members share an extended N-terminal region of low sequence homology. Sequence alignment of the N terminus of Tel1/ATM orthologs revealed a conserved, novel motif we term TAN (for Tel1/ATM N-terminal motif). Point mutations in conserved residues of the TAN motif resulted in telomere shortening, and its deletion caused the same short telomere phenotype as complete deletion of Tel1 did. Overexpressing Tel1 TAN mutants did not rescue telomere shortening. The TAN motif was also essential for the function of Tel1 in the response to DNA damage, as TAN-deleted Tel1 was indistinguishable from the complete lack of Tel1 in causing reduced viability and signaling through Rad53 upon DNA damage. Strikingly, TAN deletion reduced recruitment of Tel1 to a double-strand DNA break. Together, these results define a conserved sequence motif within an otherwise poorly defined region of the Tel1/ATM kinase family proteins that is essential for normal Tel1 function in Saccharomyces cerevisiae.     

Structure of the human dimeric ATM kinase

DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition.     

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA

DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.Key words: DNA double strand breaks, nonsense-mediated mRNA decay (NMD), oxidative stress, phosphatidylinositiol-3-kinase-like kinases (PIKKs), RNA damage     

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation,fork restart,homologous recombination and mitotic catastrophe

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

Polo-like kinases and Chk2 at the interface of DNA damage checkpoint pathways and mitotic regulation

The cell cycle controls processes of DNA replication and segregation of replicated DNA into two daughter cells. These processes are coordinated by multiple signaling pathways, which employ many protein kinases. The members of the family of Polo-like protein kinases are among these key cell cycle regulators. In response to DNA damage and inhibited DNA replication, DNA structure checkpoints delay cell cycle progression to provide cells with time for repair of damaged DNA and protect it from more severe damage. These effects are achieved by affecting key players of the basic cell cycle regulation of the cells with damaged DNA. This review is focused on the interplay between Chk2, a bona fide checkpoint protein kinase, and Polo-like kinases.