CtIP: A DNA damage response protein at the intersection of DNA metabolism

DNA Repair 32 (2015) 75–81

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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

CtIP: A DNA damage response protein at the intersection of DNA metabolism Nodar Makharashvili, Tanya T. Paull ∗ The Howard Hughes Medical Institute, The Department of Molecular Genetics and Microbiology, The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA

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Article history: Available online 2 May 2015 Keywords: Double-strand break DNA damage response CtIP Mre11/Rad50/Nbs1 Resection

a b s t r a c t The mammalian CtIP protein and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. Here we review the current literature supporting the role of CtIP in DNA end processing and the importance of CtIP endonuclease activity in DNA repair. We also examine the regulation of CtIP function by post-translational modifications, and its involvement in transcription- and replication-dependent functions through association with other protein complexes. The tumor suppressor function of CtIP likely is dependent on a combination of these roles in many aspects of DNA metabolism. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The CtBP (C-terminal binding protein) interacting protein (CtIP) was initially identified as part of the CtBP transcriptional corepressor complex that mediates repression of many genes and plays important roles in development and cancer [1,2]. In addition, CtIP was found to bind directly to the retinoblastoma (Rb) protein [3], as well as the tumor suppressor BRCA1 [4], suggesting the protein might function in tumorigenesis. Despite, an initial association with the regulation of gene expression, CtIP is now better known for its role in DNA double-strand break (DSB) repair and genome stability. CtIP is an interacting partner of the Mre11/Rad50/Nbs1 (MRN) DNA damage sensor protein complex, which recognizes DNA double-strand breaks (DSBs) and promotes the resection of 5 strands to generate 3 single-stranded intermediates that are necessary for homologous recombination [5–11]. CtIP is involved in these pathways supporting the initial steps of DNA processing as well as in the recruitment of additional DNA repair proteins

Abbreviations: CtIP, C-terminal binding protein 1 (CtBP1) interacting protein; DSB(s), double-strand break(s); The MRN complex, Mre11/Rad50/Nbs1; ATM, ataxia-telangiectasia mutated kinase; ATR, ATM and Rad3 related kinase; IR, ionizing radiation; UV, ultraviolet; MMS, methanesulfonate; CDK, cyclindependent kinase; CPT, camptothecin; HR, homologous recombination; MMEJ, microhomology-mediated end joining; NHEJ, non-homologous end joining; The 9–1–1 complex, Rad9–Hus1–Rad1; LMO4, LIM domain transcription factor 4. ∗ Corresponding author. E-mail address: [email protected] (T.T. Paull). http://dx.doi.org/10.1016/j.dnarep.2015.04.016 1568-7864/© 2015 Elsevier B.V. All rights reserved.

[10,12–14]. There are no human conditions associated with complete CtIP deficiency, as it is lethal based on deletion experiments in the mouse [15]. Rare hypomorphic mutations in human CtIP have been identified, however, which lead to severe intrauterine growth retardation, profound microcephaly, dwarfism, mental retardation, and isolated skeletal abnormalities in patients with Seckel and Jawad syndromes [16,17]. CtIP is a known tumor suppressor. CtIP heterozygosity in the mouse generates a high frequency of tumorigenesis, indicating that CtIP haploinsufficiency is linked to cancer [15]. Mutations in CtIP are also found associated with endometrial, colorectal, breast, ovarian, and myeloid cancers in humans [18–21]. A homopurine repeat in CtIP is a hotspot for 1 bp deletions in colorectal cancers, which generates a truncated form of the protein [21]. A significant link also exists between CtIP levels, certain CtIP point mutations, and specific breast cancer types, while the absence of CtIP in breast cancer cells is associated with tamoxifen resistance [22,23]. Thus, CtIP has been suggested to be an important biomarker for breast cancer prognosis and clinical management [23]. CtIP interacts with a large number of proteins related to carcinogenesis. BRCA1 is a well known gene product linked to familial ovarian and breast cancers, while other interactors, such as the Rb protein, the LIM-only protein 4, CtBP, ZBRK1, and HMGA2, are also associated with cancer and regulate different branches of DNA metabolism including replication and transcription [2,24–27]. These diverse interactions may also explain the severe effects of CtIP deletion on mammalian cells.

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Fig. 1. Schematic diagram of CtIP showing known features. Orange and grey regions depict multimerization and MRN binding domains, respectively. Rb-, BRCA1-, and CtBP-binding motifs are shown at a.a. 153–157, 327, and 490–494, respectively [1,3,47,68]. S/TP (green) and S/TQ (red) phosphorylation sites are depicted at S276, T315, S327, S347, T847, and S231, S664, S745, T859, respectively [10,39,40,44–46,49]. K432, K526, K604 acetylation sites [41] are shown in blue. 608 a.a. Jawad, and 782 a.a. Seckel 2 (shown in purple) denote sites of truncations associated with Jawad and Seckel 2 syndromes (Note: C-termini of both truncations have alterations in sequences due to open reading frame shift) [17]. Nuclease domain includes residues from approximately 180 to 350 [10,12]. The K513/K515 residues highlight residues implicated in DNA binding [117].

2. CtIP is an endonuclease The CtIP gene product is an endonuclease with specificity for 5 flaps of splayed DNA, although it binds to different DNA structures with comparable affinity [10,12]. To cleave the 5 overhang, CtIP requires both 3 and 5 flaps of a Y-DNA structure, and unlike its yeast functional homolog Sae2 it does not cleave single-stranded DNA adjacent to hairpin structures [28]. CtIP requires a divalent metal for its catalytic activity and shows the highest activity with Mn2+ ions, similar to the endo/exonuclease Mre11 [10]. However, unlike Mre11 [29], substituting Mg2+ for Mn2+ does not change either the polarity or pattern of DNA cleavage [10]. Recombinant CtIP co-purifies with an unknown transition metal, which in the presence of sodium ascorbate and hydrogen peroxide leads to cleavage of the CtIP protein in N-terminal region encompassing residues 181–290 [10]. Through limited sequence similarity searches and auto-proteolysis patterns, Makharashvili et al. and Wang et al. identified several groups of residues, N289/H290, N181/R185, and E267/E268, responsible for CtIP nuclease activity [10,12]. Recombinant CtIP protein with mutations in these residues shows severely diminished nuclease activity, but retains wild-type DNA binding ability, suggesting that these mutants can be used as separation of function alleles specific for loss of nuclease activity. Analysis of these mutants reveals that CtIP has at least two distinct roles in the processes of DSB end resection. DSBs produced by restriction enzymes create simple broken ends that require the CtIP protein but not its nuclease activity [10,12]. In contrast, inverted DNA repeats, topoisomerase poisons, and ionizing radiation (IR) result in more complex DNA lesions with extruded DNA hairpins, protein–DNA adducts, or mixed types of DNA damage, respectively, and repair of these lesions requires not only the CtIP protein but also its nuclease activity. Similar patterns of sensitivity to DNA damaging agents and protein–DNA adducts were observed in CtIP deletion mutants in other organisms, suggesting a conserved mechanism of resolution of these lesions [30–34]. Thus, CtIP nuclease activity is directly implicated in the removal of covalently linked proteins and severely damaged bases from DNA, in addition to a role for the CtIP protein in DNA repair that likely occurs through CtIP-dependent recruitment of other DNA damage repair factors (Fig. 1). 3. CtIP and its regulation in cells CtIP interacts with a large number of proteins involved in different branches of DNA metabolism, including DNA damage repair, DNA replication, and transcription regulation. The structure of the protein is not well defined; however, it is clear that the N-terminal half of CtIP and its orthologs contains domains responsible for

protein’s DNA binding and nuclease activities, as well as for multimerization and protein–protein interactions [10,12,35–38]. The C-terminus of CtIP is likely to have a regulatory function, since point mutations and partial deletions, but not its complete removal, render the enzyme inactive [10,12,39]. Despite extensive analysis of CtIP by many groups, however, a complete understanding of how CtIP is regulated remains elusive. Cell cycle- and DNA damagedependent enzymes modify CtIP by phosphorylation, acetylation, ubiquitination, and proline isomerization, which affect protein’s nuclease activity, interactions with CtIP’s partner proteins, and proteasome-mediated degradation [10,39–46]. CtIP is extensively phosphorylated by cyclin- (CDK) and DNA damage-dependent kinases, which modulate different CtIP activities [10,39,40,44–50]. For example, the T847A and S327A CtIP mutants that cannot be phosphorylated by CDK are proficient in DNA binding and nuclease activity in vitro [10], but cannot complement the sensitivity of CtIP-depleted cells to DNA damaging agents, including ionizing radiation (IR), UV, cisplatin, methyl methanesulfonate (MMS), topoisomerase 1, and topoisomerase 2 poisons [39,40,48,51]. In contrast, a mutation in CtIP serine 347, also an SP site, yields a mutant that is nuclease inactive and supports restriction enzyme induced-, but not camptothecin- (CPT), etoposide-, or IR-induced DNA damage repair [10,40]. The S276A and T315A S/TP mutations, which abolish Pin1-mediated isomerization of the P277 and P316 proline residues to target CtIP for degradation, also render the protein nuclease-deficient, and make cells sensitive to DNA damage [10,42]. The S347 and S276 site are part of a group of CDK sites identified by Wang et al. that promote the binding of Nbs1 to CtIP and also promote phosphorylation by ATM [40], thus, some of the modifications of CtIP are clearly essential for multiple functions. Interestingly, the non-cyclin dependent kinase Plk3 was shown to phosphorylate the S327 and T847 sites in the G0 /G1 cell cycle phases in response to DSBs [46], consistent with other reports suggesting roles for CtIP in G1 [52–54]. However, in the absence of HR repair machinery these modifications engage CtIP and MRN into MMEJ-mediated repair [53]. Similar diverse effects are observed with the S/TQ sites modified by DNA-damage-dependent kinases, and in some cases CDK modification is required for ATM-mediated phosphorylation [40], similar to modifications of Sae2 in budding yeast [55]. The ATM phosphorylation sites on CtIP, S664, and S745, were originally reported as modifications of CtIP that blocked interaction of Brca1 after DNA damage [44]. However, these ATM-dependent sites, along with the S231 SQ site on CtIP are also essential for CtIP nuclease activity in vitro [10]. The ATR kinase has also been shown to phosphorylate CtIP, on the DNA damage-dependent TQ site T859 in human CtIP

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and T818 in xenopus, although evidence for ATM phosphorylation of this site has also emerged [40,45]. Modification of the T859 site in human CtIP does not alter its nuclease activity, but is essential for CtIP recruitment to chromatin, full checkpoint activation, and for the non-catalytic role of CtIP in DNA repair of restriction enzymeinduced breaks [10,45]. The CDK-dependent and PI3 kinase-like kinase- (PIKK) dependent sites of phosphorylation in CtIP were also shown to be essential for the recruitment of Exo1 and BLM to sites of laser-induced damage [40]. Thus, CtIP modification by different DNA damage-dependent kinases is required for regulation of both its catalytic and non-catalytic roles. CtIP is also acetylated, ubiquitinated, and NEDDylated [41,43,56,57]. De-acetylation of K432, K526, and K602CtIP residues by Sirt6 plays a critical role in response to DNA damage by promoting recruitment of CtIP to chromatin [41]. While the triple lysine-to-arginine acetylation mutant complements DNA damage sensitivity in the absence of Sirt6, both 3K-to-3R and 3K-to-3N mutants retain wild-type in vitro nuclease activity, suggesting that this modification may primarily affect its recruitment to chromatin [10]. The APC/C(C) (dh1) ubiquitin ligase modifies CtIP by interacting through CtIP’s conserved KEN box and downregulates CtIP both during the G1 phase of the cell cycle and after DNA damage in G2 phase [57]. Phospho-dependent Pin1-mediated proline isomerization of CtIP was also shown to promote the ubiquitination of CtIP and subsequent degradation [42], and BRCA1 can also ubiquitinate CtIP in vitro; the ubiquitinated form of the protein was not degraded in this case but shown to associate more efficiently with chromatin [43]. NEDDylation has also been shown to modulate interaction between BRCA1 and CtIP, thus, altering short- and long-range resection profiles [56]; however, it is not clear whether NEDDylation does so by altering phosphorylation of the S327 site, which is implicated in CtIP–BRCA1 interaction, or by obstructing binding through another mechanism.

4. CtIP and Sae2 CtIP is a functional ortholog of the budding yeast endonuclease Sae2, although they share very limited sequence homology [6]. Both Sae2 and CtIP interact with the Mre11/Rad50/Xrs2(Nbs1) complex and are involved in the repair of DSBs [6–11]. Orthologs of these proteins are now recognized in fission yeast, plants, and nematodes and in every case they are involved in DNA damage responses and meiotic progression [14,33,34]. Functional similarities extend to their activities in DNA end processing and removal of hairpin secondary structures and protein–DNA adducts [10,12,28]. Sae2, similar to CtIP, undergoes extensive phosphorylation events, which not only depend on each other, but also regulate Sae2 nuclease and DNA repair activities [55]. Recent data indicate that CDK-mediated phosphorylation primes Sae2 for DNA damage-dependent phosphorylation by Tel1 (yeast homolog of human ATM), which triggers a transition from large oligomeric state into smaller active units. Blocking this transition by mutating CDK- and Tel1-dependent sites severely diminishes cell survival in the presence of DNA damaging agents as well as Sae2 nuclease activity [55]. However, blocking Sae2 into a monomeric state by the L25P mutation does not affect its nuclease activity but still leads to DNA damage sensitivity, meaning that transition between large and smaller species is an essential mechanism that tightly regulates Sae2 involvement in cellular processes. CtIP also exists as a multimer, but it is unknown whether it undergoes a structural transition similar to Sae2 upon DNA damage [35–37]. The N-terminus of CtIP spanning residues 45–160 forms a multi-helical compact structure and is important for its dimerization [35]. Gel filtration experiments also indicate that recombinant human CtIP exists as a wide range of multimers that include

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oligomers and dimers (N.M., unpublished observations). In addition, recent publications present the crystal structure of the CtIP N-terminus showing a tetrameric association [36,38]. This apparent contradiction may lie in the ranges of concentrations used for analysis in each study, as well as the possible elongated shape of CtIP that could elute at a larger apparent molecular mass. However, all these studies agree that mutation of the L27 residue alone or in combination with neighboring sites disrupt formation of large oligomeric species, similar to the L25P mutation of Sae2 [55,58]. CtIP mutants that do not tetramerize fail to support efficient DNA end resection and DNA damage survival in cells [36,38], suggesting that the oligomeric structure of CtIP is essential for its function. In the case of Sae2, it is clear that the L25P mutant form was not phosphorylated in response to DNA damage and was also largely degraded in vivo [55], but it remains to be seen if the oligomerization of CtIP in mammalian cells also impacts its post-translational modifications. 5. CtIP and MRN Functional interaction between the MRX complex and Sae2 was discovered through genetic analysis of Spo11-mediated meiotic DSB repair [59,60]. Sae2 null strains, similar to a set of rad50S and mre11S hypomorphic mutant strains, cannot resolve Spo11induced meiotic DSBs [30]. CtIP/Sae2 and MRN/MRX are both involved in the initial steps of DSB end processing in eukaryotic cells [61,62], and in vitro they also promote formation of long 3 singlestranded DNA intermediates by the long-range exonucleases Exo1 and Dna2 [10,13,63–65]. The in vitro results with purified proteins show that both Sae2 and CtIP promote interactions between Exo1/Dna2 and DNA ends, particularly in the presence of the Ku heterodimer which creates a strong block to resection [10,13,63]. These activities likely constitute at least part of the non-catalytic role of Sae2 and CtIP in DNA end resection. Physical interactions between MRX/N and Sae2/CtIP also occur [6,7,9–11], and a crystal structure of the N-terminus of Schizosaccharomyces pombe Nbs1 bound to a phospho-peptide from the Ctp1 N-terminus has been solved [8]. With budding yeast Sae2, interactions with MRX are not observed with Sae2 purified from Escherichia coli but Sae2 purified from insect cells exhibits physical interactions with MRX, consistent with the interpretation that interaction between these two complexes is also mediated by phosphorylation of Sae2 [66]. Interaction between human CtIP and the MRN complex is facilitated by the FHA and BRCT phospho-peptide binding domains of Nbs1, and phosphorylation of 5 of 12 putative CDK sites of CtIP [40]. Similar to phosphorylation of CDK sites on Sae2 [55], these modifications also prime CtIP for ATM-dependent phosphorylation [40]. Mutation of the critical CDK sites compromise phosphorylation of the T847 ATM- and T859-ATR sites, rendering the protein inactive in HR reporter assays. Even though the CtIP(T847A) and CtIP(T859A) mutants do not support HR, they are nuclease proficient in vitro, and capable of binding to Nbs1, underlining the fact that these modifications affect a general CtIP-dependent resection function independent of its nuclease activity [10,40]. 6. CtIP and BRCA1 The exact role of BRCA1 in DNA damage repair is not well defined. The BRCT domain of BRCA1 binds a large number of proteins through their phosphorylated serine residues, which may suggest BRCA1 functions primarily as a regulatory protein [67]. The BRCA1 interaction with CtIP depends on phosphorylation of S327 residue of CtIP [47,68]. Chicken DT40 cells expressing a S332A mutant CtIP allele, an equivalent of the S327A mutation of human

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CtIP, show defective CtIP-mediated homologous recombination (HR) and single-strand annealing (SSA), yet still functional nonhomologous end joining (NHEJ), and microhomology mediated end joining (MMEJ) repair pathways in response to IR-induced DNA damage, suggesting that BRCA1 has an important role in regulation of the DNA damage repair pathway choice in DT40 cells [52]. However, another study of the S332A mutation in DT40 demonstrated that the mutation yields wild-type levels of HR despite sensitivity to the topoisomerase poisons camptothecin and etoposide, and that a complete deletion of CtIP in these cells blocks cellular proliferation [48]. The discrepancies between these studies have not been resolved and results from mammalian models provide yet another view of this controversy. Data from a study describing a mouse expressing S327A knock-in mutation in CtIP indicates that the BRCA1–CtIP interaction only mildly affects resection rate, and that the mice carrying mutant alleles do not have elevated susceptibility to cancer [51]. Moreover, this mutation does not detectably affect resection, maintenance of genomic stability or viability in mouse cells. While these findings are not in agreement, it is possible that discrepancies might be explained by additional interactions between the mammalian proteins that can partially rescue lost interaction at the S327CtIP site. Antagonistic activities of BRCA1 and 53BP1 are thought to dictate the choice of DSB repair depending on the cell cycle phase [69–74]. It was proposed that BRCA1 potentiates MRN-dependent resection to compete with Ku-dependent NHEJ by stimulating resection while 53BP1 suppresses resection through RIF1 and the Ku complex. CtIP and BRCA1 clearly have distinct roles in this regard. Although 53BP1 deletion rescues BRCA1−/− lethality in early development, it does not rescue CtIP−/− lethality or DNA damage repair deficiency [51], indicating that CtIP provides a unique function. Depletion of BRCA1 does not affect the efficiency of DNA resection or the number of RPA foci, even though the number of Rad51 foci is reduced, and cell survival is compromised [75]. A recent report shows that BRCA1 binding to CtIP and MRN can modestly promote DSB resection and formation of a 3 single-strand DNA overhang [76], but it is not clear how such a small reduction can be responsible for BRCA1−/− lethality and DNA damage sensitivity. It is evident that BRCA1 has both CtIP-dependent and independent functions, but the exact roles of BRCA1 in DNA damage still remain elusive. BRCA1 also forms complexes with transcription factors and RNA polymerase [77–79], and has been shown to be important for the resolution of stalled replication forks [80,81]. Thus, the interaction of BRCA1 with CtIP is likely only one part of its complex set of roles in DNA damage repair.

7. CtIP and topoisomerases As processive DNA and RNA polymerases translocate along dsDNA, they create positive and negative supercoiling of the double helix, which is removed by topoisomerases [82,83]. Topoisomerase inhibitors and poisons disrupt the full cycle of these enzymes, creating DNA breaks with terminal topoisomerase adducts [84–86]. Tyrosyl-DNA phosphodiesterases (Tdp1 and -2) remove trapped topoisomerases that have been partially processed by the proteasome, generating clean end DSBs [87]. CtIP also plays a significant role in the resolution of topoisomerase adducts as CtIP deficient cells exhibit marked sensitivity to topoisomerase poisons [6]. Moreover, nuclease-dead CtIP, which is capable of supporting the repair of clean DSBs, does not rescue sensitivity to these poisons, suggesting that there is a subset of topoisomerase adducts that require the CtIP nuclease-dependent pathway [10,12]. The CtIPdependent pathway is parallel to the Tdp1-mediated pathway of

topoisomerase adduct processing, based on experiments in chicken DT40 cells [88]. DNA damage response proteins associated with CtIP also play important roles in topoisomerase break repair. For example, ATMdeficient neuronal cells are prone to accumulation of spontaneous topoisomerase-DNA adducts [89], which could be due to the requirement of ATM phosphorylation for CtIP nuclease activity [10]. ATM also interacts with and phosphorylates TopBP1 protein that binds to topoisomerase II, recruiting MRN to damage sites, suggesting that deficiency in MRN recruitment could result in similar sensitivity [90]. MRN was also shown to contribute to the recruitment of TopBP1 to ATR-activating structures and to promote ATR activation [91], providing an additional link between MRN/CtIP and replication. Topoisomerase inhibitors are a successful class of anti-cancer drugs. However, severe side effects accompany topoisomerase II inhibitors and they have a limited usage despite huge potential [92,93]. Topoisomerase I drugs are better tolerated and are widely used [94]. While the search for better topoisomerase drugs is ongoing, CtIP-mediated repair pathways may be a source of more specific and less toxic targets that repair topoisomerase lesions in human cancers.

8. CtIP and replication In eukaryotes, DNA damage encountered during replication and the G2 phase of the cell cycle is predominantly repaired via homologous recombination [95–97]. Stressed or stalled replications forks that lead to single-strand gaps activate ATR (ataxia telangiectasia and Rad3 related) kinase [98]. Optimal ATR activation requires at least two events: the recruitment of the Rad9–Hus1–Rad1 (9–1–1) complex and the binding of the ATR–ATRIP complex to RPA [99]. The CtIP-associated genetic disorders have a very similar phenotype to the disorders associated with the ATR hypomorphic mutations, and CtIP is also phosphorylated by ATR [45], which suggests an epistatic interaction between these two gene products [17,100]. CtIP is likely involved in the processing of DNA intermediates that arise during replication at sites of secondary structures as well as in the initiation of resection of DSBs formed during this process [10,12]. Specifically, CtIP promotes removal of secondary DNA structures, protein–DNA adducts, as well as interstrand DNA crosslinks, all of which act as replication fork blocks. This is conceptually similar to the proposed role of the Mre11/Rad50 complex, which also contributes to the removal of hairpin structures at sites of inverted repeats in prokaryotes and eukaryotes [101–104]. Many lines of evidence also show that CtIP is critical for the resolution of removal of covalently linked topoisomerases from DNA through a process that involves its nuclease activity. Moreover, CtIP interacts with the FANC protein complex in replication-dependent repair of DNA crosslinks by directly binding FANCJ, FANCM, and BRCA1 components of the FANC complex and activating DNA damage response kinases [105–107]. Besides its DNA repair activities, CtIP may be involved directly in regulation of replication as it interacts with Rb, a major cell cycle regulator, and p130 proteins through its Rb-binding motif, LXCXE [3,108–111]. When hyperphosphorylated, Rb allows cells to progress into S-phase, and cells with dysfunctional or absent Rb can become transformed. The absence of CtIP prevents hyperphosphorylation of Rb and cell cycle progression into the S phase, which could be one reason why CtIP deficiency is so deleterious for cells [15]. The mechanism by which CtIP modulates Rb activities is not clear, but it could be by CtIP-mediated promotion of p21 expression, as was proposed recently [112].

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9. CtIP and transcription regulation CtIP was originally identified as a transcription regulator [3,108,113], and is known to interact with the transcription repressors CtBP, LMO4 (LIM domain transcription factor 4), and Ikaros, as well as transcription co-regulators E2F and TFIIB [1,114–116]. However, what roles CtIP plays in gene regulation and how this relates to its DNA damage repair properties remains unknown. It is possible that CtIP acts in the resolution of DNA lesions encountered during transcription, but this hypothesis is unproven and does not explain the effect of CtIP on a specific subset of genes including p21 and the CtIP itself [114]. In this regard, a much better understanding of the function of CtIP interactions with transcription-related factors will be needed, especially considering that many of these proteins are also linked to various types of cancers.

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10. Concluding remarks A large body of evidence suggests that CtIP efficiently coordinates DNA damage repair, DNA replication, and RNA transcription through its interactions with diverse families of proteins. Its role is unique in this regard and can provide a great opportunity to develop therapeutics targeting the processes it is involved in. Since CtIP is required for the efficient repair of topoisomerase-DNA adducts caused by a widely used class of anti-cancer topoisomerase drugs, modulation of CtIP nuclease activity or its interacting proteins can provide a means to further sensitize DNA damage repair deficient tumor cells to these drugs. To accomplish this and also to better understand the roles of CtIP as a tumor suppressor, we need to clarify the specificity and regulation of the nuclease activity of CtIP, how this relates mechanistically to the nuclease activities of Mre11, and to define how its functions relate to the other known pathways of DNA–protein adduct repair and DNA end resection.

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