Proteins involved in DSB end resection.
1. Introduction
Double-strand breaks (DSBs) are among the most serious forms of DNA damage and thus must be efficiently repaired. In order for effective repair to take place, cells must employ the following steps: 1) recognition of the DSBs, 2) cell-cycle arrest via checkpoint activation, and 3) repair of the breaks. In the repair step, DSB ends are nucleolytically processed, which leads to the subsequent recruitment of appropriate repair proteins. Several proteins, including the Mre11 nuclease, are known to be involved in the processing of DSB ends. Additionally, recent studies have identified human CtIP and its orthologs as novel components required for DNA end processing among eukaryotes. This protein is involved not only in repair via homologous recombination (HR) but also in several important biological processes, such as transcriptional regulation and checkpoint control. Importantly, CtIP acts as a tumor suppressor in mammals. In this chapter, we will summarize the existing knowledge on this multi-functional molecule.
2. Identification of CtIP
2.1. DSB repair pathways
DSBs could lead to chromosomal aberrations, the disruption of genome integrity, and tumorigenesis in higher eukaryotes. DSBs are generated either by exogenous sources such as gamma-irradiation or by endogenous factors such as replication fork collapse. In addition, programmed DSBs are induced during meiosis at several loci, known as recombination hot spots. While such programmed DSBs are repaired by error-free HR, incidentally-generated DSBs are repaired by one of two major DNA repair pathways: HR or error-prone non-homologous end-joining (NHEJ), both of which are highly conserved from yeast to humans. NHEJ and HR function predominantly in cell cycle phases G1 and S/G2, respectively. DSBs are repaired using intact homologous sequences (sister-chromatids or homologs) as a template in HR, whereas in NHEJ, the broken ends are directly rejoined.
When DSBs occur, a protein complex called MRN(X) (see below for details) is recruited at the DSB ends and activates the DNA damage checkpoint. The Ku70-Ku80 heterodimer, involved in the NHEJ pathway, is also recruited. The ends are then nucleolytically processed by the MRN(X) complex in a process called “DNA end resection” or simply “resection”, leading to the conversion of “dirty ends” to repairable “clean ends”. Human CtIP and its orthologs, together with the MRN(X) complex, play a critical role in this DNA end processing.
2.2. Several Y2H screenings with different ‘baits’ identified CtIP
Human CtIP was first identified by a yeast two-hybrid (Y2H) assay as one of the interacting proteins of CtBP (C terminus-binding protein), which is a transcriptional corepressor (Schaeper et al., 1998). CtBP binds to the C-terminal PLDLS motif of adenovirus E1A, resulting in anti-tumorigenic activity (Schaeper et al., 1995; Boyd et al., 1993). CtIP was also identified as RBBP8 in another Y2H screen in which the retinoblastoma (Rb) protein, a tumor suppressor with a protein-binding “pocket” domain, was applied as a bait (Fusco et al., 1998). CtIP/RBBP8 contains the LECEE sequence, known as an Rb-binding domain, which is required for interaction with Rb in a Y2H system (Fusco et al., 1998). CtIP was also found to associate with Rb-related protein p130 in a different Y2H screening (Meloni et al., 1999).
The breast and ovarian tumor suppressor BRCA1 has important functions in cell cycle checkpoint control and DNA repair. Two tandem BRCA1 C-terminal (BRCT) motifs are essential for the tumor suppression activity of BRCA1. The BRCT motifs of BRCA1 have also been shown to interact with CtIP both
CtIP has also been isolated in two independent Y2H assays with Ikaros and LMO4 used as bait proteins (Koipally and Georgopoulos, 2002; Sum et al., 2002). Ikaros is a zinc finger protein that plays key roles in hemolymphoid development and homeostasis (Koipally and Georgopoulos, 2002). LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators (Sum et al., 2002).
2.3. Identification of CtIP homologs
Sae2/Com1 was identified in the budding yeast
SpCtp1 was also found to be coded by an
CeCom-1 was originally identified from a mutagenesis screen for mutants causing maternal-effect embryonic lethality (Penkner et al., 2007; Gönczy et al., 1999). AtGR1 was isolated from a screen for mRNAs that accumulate after DNA damage induced by ionizing radiation (Deveaux et al., 2000).
2.4. CtIP is conserved protein from yeast to humans
As previously mentioned,
not since been reported in any organism due to limited sequence similarity. The recent identification of SpCtp1 has led to the insight that it is homologous to proteins that have previously been characterized in several species such as ScSae2/Com1, CeCOM-1, AtGR1, and human CtIP (Limbo et al., 2007). In parallel, Jackson’s group suggested that human CtIP shares some sequence homology with ScSae2 (Sartori et al., 2007). CtIP family proteins share several domains with highly conserved sequences but also demonstrate highly diverse sequences in other regions and show variety in protein length. These features might clarify why the orthologs were not recognized for a long time.
2.5. Domain structure of CtIP and its homologs
CtIP homologs retain C-terminal core domains, including RHR and CxxC motifs (Fig. 1A). The RHR motif is proposed to be the representative signature of Sae2/Com1 homologs (Limbo et al., 2007) and the limited 30 aa-region containing the RHR motif is well-conserved from ScSae2/Com1 to human CtIP (Fig. 1B). The CxxC motif is generally found in proteins with a D-box (RxxL) motif, which is representative among APC/C substrates, and the CxxC motif in some proteins is known to be involved in zinc chelation (Hopfner et al., 2002). The CxxC and D-box motifs are conserved among CtIP homologs from fission yeast to humans with the exception of some other fungi, including
2.6. Posttranslational modifications of CtIP and its homologs
Several post-translational modification sites have been identified in CtIP homologs. Human CtIP has two CDK-dependent and two ATM-dependent phosphorylation sites. Phosphorylation at Ser-327 of CtIP by CDK increases around S/G2 phases in unperturbed cells and might be responsible for CtIP-BRCA1 complex formation, which occurs in G2 phase (Yu and Chen, 2004). Although this phosphorylation site does not seem to be conserved among species, another CDK-dependent site, Thr-847, which is likely to be conserved from yeast to humans, has been identified (Huertas and Jackson, 2009). Two ATM-target sites, Ser-664 and Ser-745, are phosphorylated in response to DNA damage, leading to Chk1 phosphorylation and the G2/M transition checkpoint (Li et al., 2000). In addition to these phosphorylation events, CtIP is ubiquitinylated by the BRCA1-BARD1 heterodimer
Similar to what has been observed in human CtIP, ScSae2 is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage (Baroni et al., 2004). Both cell cycle- and DNA damage-dependent Sae2 phosphorylation require the checkpoint kinase Mec1. Another pathway, involving Tel1 and MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation (Baroni et al., 2004). Sae2 contains three potential CDK phosphorylation sites, Ser-134, Ser-179 and Ser-267, the last of which is a well-conserved residue that maps to the C-terminal region most highly conserved among organisms (Fig. 1C). Mutation of Ser-267 to an Ala residue causes phenotypes comparable to those observed in the
In
3. CtIP and its homologs are involved in several biological processes
3.1. CtIP is involved in transcriptional regulation
As mentioned above, CtIP interacts with several proteins involved in transcriptional regulation, one of which is CtBP. CtBP acts as a transcriptional corepressor of several tumor suppressors such as E-cadherin, p16Ink4a, p15Ink4b, and PTEN, indicating a strong association with tumorigenesis and tumor progression (Chinnadurai, 2009). Complete transcriptional repression by CtIP requires binding to CtBP through its PLDLS domain (Meloni et al., 1999). The PLDLS motif of adenovirus E1A disrupts the CtBP-CtIP complex
CtIP is also suggested to be a corepressor with Rb and p130 (Meloni et al., 1999). In contrast, CtIP has also been shown to bind Rb, allowing CtIP to bind its own promoter and an E2F target such as cyclin D1 during the G1/S transition (Liu and Lee, 2006). This releases Rb-mediated transcriptional repression and increases the expression of genes required for S-phase entry. Furthermore, other groups have shown that CtIP can interact with the general transcription factors, TATA binding protein (TBP) and transcription factor IIB (Koipally and Georgopoulos, 2002). However, the functions of CtIP in transcriptional regulation might be limited to vertebrates and its orthologs might not play an important role in transcription.
3.2. Meiotic recombination and HR repair
In meiosis of
During mitosis,
Sc | Sp | Hs | Note |
Mre11 | Rad32 | MRE11 | single-strand endonuclease 3’-5’ double-strand exonuclease weak hairpin-opening activity |
Rad50 | Rad50 | RAD50 | split ABC-type ATPase containing two heptad repeats stimulates the 3’-5’ exonuclease and hairpin-opening activities of Mre11 |
Xrs2 | Nbs1 | NBS1 | contains an N-terminal FHA domain and a small C-terminal conserved domain Nbs1, but not Xrs2, contains a BRCT domain in the N-terminal region overall sequence similarity between Xrs2 and Nbs1 is weak and limited to an N-terminal FHA domain and a small C-terminal conserved domain |
Sae2 | Ctp1 | CtIP | endonuclease activity on single stranded DNA (Sae2) stimulates nuclease activity of MR complex (Sae2 and CtIP) |
Exo1 | Exo1 | EXO1 | 5’-3’ exonuclease, flap endonuclease |
Sgs1 | Rqh1 | BLM | RecQ family DNA helicase |
Rmi1 | Rmi1 | RMI1 (BLAP75) | RecQ-mediated genome instability protein forms a complex with Sgs1 and Top3 |
Top3 | Top3 | TOPOIIIα | type 1A topoisomerase |
Dna2 | Dna2 | DNA2 | 5’-3’ helicase/endonuclease |
Sgs1, a RecQ family helicase, forms a protein complex called RTR with Top3 and Rmi1. The RTR complex has multiple functions in DSB repair, including double Holliday junction dissolution (for review see (Ashton and Hickson, 2010)). The MRX complex and Sae2 in
Biochemically, Mre11 retains 3’->5’ exonuclease and ssDNA endonuclease and hairpin opening activities, all of which require Mn2+ as a metal cofactor (Trujillo and Sung, 2001; Sigurdsson et al., 2001). Both ATP and Rad50 stimulate the 3’->5’ exonuclease and hairpin opening activities of Mre11, where ATP is thought to regulate the DNA binding of the Mre11 complex via Rad50 (Trujillo and Sung, 2001). Remarkably, the ATP-dependent DNA end-resection reaction including the MRX complex, the RTR complex, Dna2 and the heterotrimeric ssDNA-binding protein RPA has been reconstituted (Cejka et al., 2010; Niu et al., 2010). Sae2 itself is shown to stimulate the nuclease activity of Mre11 and, interestingly, can cleave hairpin DNA, even in the absence of the MRX complex, at a cleavage site on the 3’ overhang adjacent to the hairpin (Lengsfeld et al., 2007). Recently, Paull and colleagues showed that MRX and Sae2 cooperatively promote Exo1-mediated 5’ strand degradation at DNA ends
3.3. CtIP is involved in alternative NHEJ (A-NHEJ)
There are two varieties of NHEJ: classical-NHEJ (C-NHEJ) and alternative-NHEJ (A-NHEJ) (Zha et al., 2009). CtIP is required not only for HR repair in S/G2 phase but also for A-NHEJ in G1 upon generation of DSBs in human cells. The function of CtIP in A-NHEJ is independent of the phosphorylation of Ser-327 and recruitment of BRCA1. Cells expressing CtIP protein carrying mutations at Ser-327 are specifically defective in homologous recombination and show decreased levels of ssDNA after DNA damage, whereas A-NHEJ remains unaffected. Therefore, the phosphorylation of Ser-327 of CtIP is proposed to be a molecular switch to shift the balance of DSB repair from error-prone DNA end-joining to error-free homologous recombination in humans (Yun and Hiom, 2009).
3.4. Checkpoint control by CtIP-BRCA1 interaction
CtIP interacts with the BRCT motifs of BRCA1 in a manner dependent on the phosphorylation of Ser-327. The knockdown experiment shows that this interaction is required for DNA-damage-induced Chk1 phosphorylation and the G2/M transition checkpoint but not the damage-induced G2 accumulation checkpoint, for which BRCA1-BACH1 interaction is required. Therefore, the BRCA1 checkpoint pathway is divided into the BACH1-dependent pathway and the CtIP pathway (Yu and Chen, 2004). A crystal structure of the BRCT repeats in BRCA1 with a phosphopeptide corresponding to 322–333 residues of human CtIP has been solved (Varma et al., 2005). The BRCA1-CtIP interaction is ablated by several tumor-associated mutations affecting the BRCT motifs, suggesting that the interaction may be required for tumor suppression by BRCA1 (Liu et al., 1999; Wong et al., 1998; Yu et al., 1998). On the other hand, a sequence-based screen for mutations in the CtIP coding region in a panel of 89 tumor cell line cDNAs identified five missense variants (Wong et al., 1998). Therefore, CtIP itself may act as a tumor suppressor in human cells.
The MRN complex senses DSBs and activates the ataxia-telangiectasia mutated (ATM) kinase, resulting in a DNA damage response in human cells (Lee and Paull, 2004; Uziel et al., 2003). Similarly, the MRN(X) complex is required for the activation of the ATM ortholog, Tel1, in both
Interestingly, Sae2 has been shown to be required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage in
4. Functional regulation of CtIP and its homologs
4.1. Interaction with Nbs1 regulates CtIP
The N-terminal region of Nbs1 contains FHA and BRCT motifs, both of which are known to be phosphopeptide-binding protein modules. Recently, the crystal structure of SpNbs1 has revealed that the FHA domain of Nbs1 is fused directly to the tandem BRCT domain, leading to consideration of the functional interactions of CtIP with Mre11-Rad50 through Nbs1 (Lloyd et al., 2009; Williams et al., 2008).
SpCtp1 was shown to genetically interact with SpNbs1 in
The FHA domain of hNBS1 interacts with phosphorylated SDT sites on hMDC1, whose sequence is a recognition motif for CK2 (Chapman and Jackson, 2008; Melander et al., 2008; Spycher et al., 2008). The FHA domain of SpNbs1, which is also important for cellular survival upon treatment with DNA damaging agents, is essential for Ctp1-binding
The interface of the association of Nbs1 with Mre11 maps to the C-terminal region in Nbs1 (You et al., 2005; Falck et al., 2005) and the interface of Mre11 dimerization is located away from the DNA-binding cleft (Williams et al., 2008).
The distance from the Mre11 binding domain to the N-terminal FHA domain was determined to be ~175 Å, as assessed by SAXS and X-ray crystallography (Lloyd et al., 2009; Williams et al., 2008). The MRN complex and Ctp1 colocalize to within ~200 bp (<700 Å) of a single DSB site cleaved by a defined endonuclease
4.2. CtIP homologs are highly regulated in the cell cycle
Gene expression of human and mouse CtIP proteins increases during G1 to S phase (Liu and Lee, 2006). SpCtp1 is periodically transcribed in S phase and is regarded as one of the putative MBF-regulated genes (Limbo et al., 2007). This role in transcriptional regulation and the other functions of CtIP and its homologs seem to be regulated posttranslationally. As previously mentioned, the mechanism regulating homologous recombination by CDK is conserved between human and budding yeast cells; however, the corresponding CDK phosphorylation site has not been found in SpCtp1. The regulation of Ctp1 by CDK might not be absolutely necessary as G1 phase is relatively short in
Recently, it has been found that CtIP and Sae2 are also modified by acetylation. Jackson’s group found that CtIP is constitutively acetylated but is deacetylated by SIRT6 upon treatment with a DNA damaging agent (Kaidi et al., 2010). SIRT6 is an NAD+ (nicotinamide adenine dinucleotide)-dependent sirtuin (class III), a member of the family of protein lysin deacetylases (KDACs). CtIP was identified as a target protein of SIRT6 and it was shown that deacetylation of CtIP by SIRT6 is an important regulatory event in DSB end processing.
On the other hand, Foiani‘s group revealed that treatment with the HDAC inhibitor, valproic acid (VPA), causes DSB processing defects in
5. Conclusion
As DSBs can ultimately have toxic effects on cells, such as chromosome translocation, deletion, or duplication, they must be repaired appropriately. Cells utilize several damage responses depending on the cell-cycle phase. CtIP (and its orthologs) is one of the most important key players in the initial steps of DSB repair, in which cells determine the appropriate repair pathway and process the DSB ends. In addition, the importance of posttranslational modifications of CtIP is now being elucidated. However, the precise molecular mechanism of the generation of the recombinogenic 3’ ssDNA overhang by the combined actions of CtIP/Ctp1/Sae2 and the MRN(X) complex still remains unclear.
CtIP and many of its binding partners, such as BRCA1, CtBP and Rb, are classified as tumor suppressors. Furthermore, NBS1 and MRE11 are associated with Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, both of which are chromosome instability syndromes. Cells carrying mutations in either of these genes show DNA damage hyper-sensitivity (Carney et al., 1998; Stewart et al., 1999). Taken together, these facts illustrate not only the scientific interest but also the clinical importance of understanding the molecular mechanism of DSB end resection mediated by MRN(X) and CtIP (or its orthologs).
Acknowledgments
Our study was supported by Grants-in-Aid for Scientific Research on Priority Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and for Scientific Research (A).
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