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Role of Rap1 in DNA damage response: implications in stem cell homeostasis and cancer

  • Ekta Khattar
    Correspondence
    Offprint requests to: Ekta Khattar, SVKM's NMIMS (Deemed to Be) University, Sunandan Divatia School of Science, Vile Parle West, Mumbai, Maharashtra 400056, India
    Affiliations
    Sunandan Divatia School of Science, SVKM's NMIMS (Deemed to be) University, Mumbai, India
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  • Vinay Tergaonkar
    Affiliations
    Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A-STAR), Singapore
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Open ArchivePublished:August 25, 2020DOI:https://doi.org/10.1016/j.exphem.2020.08.009

      Highlights

      • Mammalian Rap1 protein acts as an adaptor in functional complexes formed under various physiological conditions.
      • Rap1 plays an important role in the DNA damage response at telomeric and nontelomeric regions, thus maintaining genome stability.
      • The role of Rap1 in the DNA damage response has implications for stem cell survival, oncogenesis, and chemotherapeutic response.
      Mammalian Rap1 is a part of the telomere binding complex named shelterin and is one of the most conserved telomeric proteins. With its essential requirement in lower species to its becoming necessary in higher species, it appears to have gained and lost several functions simultaneously evolving with telomeres. Mammalian Rap1 has been reported to play a role in inflammation, metabolism, and oxidative stress. Mammalian Rap1 has also been found to regulate DNA damage response from telomeres in senescent cells. Recently our group uncovered its novel role in stem cell maintenance, and modulation of the chemotherapeutic response. Mechanistically it was found to function as an adaptor via protein–protein interactions and to modulate the response to DNA damage. In the current review we highlight newly identified functions of Rap1 in regulating telomeric and general DNA damage response with its impact at the cellular and organismal levels.
      Telomeres are the ends of linear chromosomes composed of tandem TTAGGG repeats in mammals, including humans. With every cell division, the telomere shortens because of the inability of canonical DNA replication to duplicate the ends of linear DNA. To overcome this end replication problem, eukaryotes employ a special enzyme named telomerase, which utilizes its reverse transcriptase activity to lengthen the telomeres [
      • Greider CW
      • Blackburn EH
      Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.
      ].
      Structurally, telomeres appear as double-strand breaks and can potentially activate DNA damage signaling and DNA repair reactions. The major enzymes involved in this DNA damage signaling include ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) kinases, DNA-dependent protein kinases (DNA-PKs), and poly(ADP-ribose) polymerase 1 (PARP1) [
      • Denchi EL
      • de Lange T
      Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1.
      ,
      • Karlseder J
      • Hoke K
      • Mirzoeva OK
      • et al.
      The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.
      ,
      • Bailey SM
      • Brenneman MA
      • Halbrook J
      • Nickoloff JA
      • Ullrich RL
      • Goodwin EH
      The kinase activity of DNA-PK is required to protect mammalian telomeres.
      ,
      • Salvati E
      • Scarsella M
      • Porru M
      • et al.
      PARP1 is activated at telomeres upon G4 stabilization: possible target for telomere-based therapy.
      ]. The DNA repair reactions involve three pathways, namely, classic nonhomologous end joining (c-NHEJ), alternative (alt)-NHEJ, and homology-directed repair (HDR) [
      • de Lange T
      Shelterin-mediated telomere protection.
      ]. To overcome this, telomeres fold into special conformations referred to as t-loops [
      • Griffith JD
      • Comeau L
      • Rosenfield S
      • et al.
      Mammalian telomeres end in a large duplex loop.
      ]. However, activation of these signaling pathways and repair cascade can result in cell cycle arrest, telomere fusions, t-loop cleavage, telomere sister chromatid exchanges (T-SCE), hyper-resection at telomeres, or telomere loss subsequently resulting in genome instability [
      • Feldser DM
      • Hackett JA
      • Greider CW
      Telomere dysfunction and the initiation of genome instability.
      ]. This is also referred to as an end protection problem encountered by telomeres. To avert this situation and maintain genome homeostasis, telomeres are associated with multiple copies of a six-membered complex named shelterin. Individual components of shelterin function in managing different aspects of these DNA damage signaling and repair reactions. Shelterin components have also been found to regulate telomere length. The shelterin complex comprises telomeric repeat-binding factor 1 (TRF1), telomeric repeat-binding factor 2 (TRF2), protection of telomeres 1 (POT1), TRF1-interacting nuclear protein 2 (TIN2), TIN2- and POT1-organizing protein (TPP1), and repressor-activator protein 1 (Rap1).
      Human Rap1 was identified as an orthologue of yeast telomere-binding protein, namely, scRap1p [
      • Li B
      • Oestreich S
      • de Lange T
      Identification of human Rap1: implications for telomere evolution.
      ]. The major difference between them is that yeast RAP1 can directly associate with telomeric DNA; however, mammalian Rap1 is recruited to telomeres via TRF2. While deletion of all shelterin components has been found to be embryonic lethal in mouse, Rap1 is an exception [
      • de Lange T
      Shelterin-mediated telomere protection.
      ]. Deletion of Rap1 in the mouse has no effect on development and normal functioning of the mouse [
      • Sfeir A
      • Kabir S
      • van Overbeek M
      • Celli GB
      • de Lange T
      Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.
      ]. Additionally, in human cell lines, Rap1 deletion has no apparent phenotype of telomere dysfunction [
      • Kabir S
      • Hockemeyer D
      • de Lange T
      TALEN gene knockouts reveal no requirement for the conserved human shelterin protein Rap1 in telomere protection and length regulation.
      ].
      Interestingly, Rap1 is an active player in regulating the DNA damage response under specific conditions, and we discuss this function of Rap1 in the light of already known data with recent findings. We have organized the review into four sections. In the first section we describe the structure of human Rap1 protein as it is important to understand and connect functions of Rap1 with its structural domains. In the second and third sections, we present the current understanding of the role of Rap1 in the DNA damage response at telomeres and away from telomeres, respectively. In the last section, we state our conclusions and future perspectives.

      Structure of Rap1 protein

      Human Rap1 is encoded by a gene named TERF2IP and was identified with a yeast two-hybrid screen of Hela cells (human cervical cancer cell line) cDNA using TRF2 as a bait protein [
      • Li B
      • Oestreich S
      • de Lange T
      Identification of human Rap1: implications for telomere evolution.
      ]. Rap1 is a 399–amino acid–long protein with a molecular weight of about 47 kDa. Rap1 protein possesses three major domains: a breast cancer susceptibility protein that appears on its C-terminus (BRCT) in the N-terminus, one central Myb motif, and a Rap1-specific protein-interaction domain at the C-terminus (RCT). Between the Myb and RCT domains lies an acidic region also called the coil–coil region whose functional significance is unclear and is more specific to humans [
      • Li B
      • Oestreich S
      • de Lange T
      Identification of human Rap1: implications for telomere evolution.
      ]. Figure 1 is a schematic of the structure of the Rap1 protein.
      Figure 1
      Figure 1Schematic of the structure of the human Rap1 protein. The domains of Rap1 with amino acid (aa) positions are indicated: breast cancer susceptibility protein that appears on the C-terminus (BRCT), Myb motif, acidic coil–coil domain, and Rap1-specific protein-interaction domain (RCT).
      The BRCT domain is normally present in various DNA damage response proteins with function in cell cycle checkpoint; in general, it interacts with phosphorylated peptides [
      • Bork P
      • Hofmann K
      • Bucher P
      • Neuwald AF
      • Altschul SF
      • Koonin EV
      A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins.
      ,
      • Leung CC
      • Glover JN
      BRCT domains: easy as one, two, three.
      ]. Deletion of this domain from human Rap1 has been reported to reduce telomere length heterogeneity [
      • Li B
      • de Lange T
      Rap1 affects the length and heterogeneity of human telomeres.
      ]. Each Rap1 molecule associates with each TRF2 molecule and, in a shelterin complex such as TRF2, is present as a dimer, two molecules of Rap1 in tandem. By use of a small-angle X-ray scattering technique, it has been observed that two Rap1 BRCT domains coming from two molecules of Rap1 are closely packed in the TRF2–Rap1 complex [
      • Gaullier G
      • Miron S
      • Pisano S
      • et al.
      A higher-order entity formed by the flexible assembly of RAP1 with TRF2.
      ]. Thus, some phospho-proteins are expected to interact with this structurally tandem BRCT domains. However, its exact function is unclear and interacting proteins have not been reported.
      The canonical Myb domain is a DNA binding motif with three alpha helices arranged in an orthogonal bundle [
      • Ogata K
      • Kanei-Ishii C
      • Sasaki M
      • et al.
      The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.
      ]. Rap1 possesses one Myb domain which has been shown to lack DNA binding activity due to lack of significant positively charged residues on its surface. It could possibly interact with DNA with the help of another protein which can provide this positive charge or it is presumed to function in protein-protein interactions [
      • Hanaoka S
      • Nagadoi A
      • Yoshimura S
      • et al.
      NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.
      ]. However, its binding partners have not been reported.
      At C-terminus Rap1 contains RCT domain, which represents a well-known protein–protein interacting domain. Rap1 interacts with TRF2 through this domain, which thus tethers it to the telomeres [
      • Chen Y
      • Rai R
      • Zhou ZR
      • et al.
      A conserved motif within RAP1 has diversified roles in telomere protection and regulation in different organisms.
      ].
      With several interaction domains, Rap1 appears to be functioning more as an adaptor introducing various factors into functional complexes at or away from telomeres [
      • Yeung F
      • Ramirez CM
      • Mateos-Gomez PA
      • et al.
      Nontelomeric role for Rap1 in regulating metabolism and protecting against obesity.
      ,
      • Teo H
      • Ghosh S
      • Luesch H
      • et al.
      Telomere-independent Rap1 is an IKK adaptor and regulates NF-kappaB-dependent gene expression.
      ]. The first mass spectrometry (MS) analysis to determine the interacting partners of human Rap1 was performed by O'Connor et al. [
      • O'Connor MS
      • Safari A
      • Liu D
      • Qin J
      • Songyang Z
      The human Rap1 protein complex and modulation of telomere length.
      ], who reported that Rap1 could associate with various DNA damage response proteins. Interestingly, they found that Rap1 could associate with Rad50/Mre-11 and Ku86 independent of TRF2, while PARP1 interaction with Rap1 required TRF2. The second MS analysis of human Rap1 was reported by our group, who also observed that Rap1 could associate with DNA damage response proteins and found that some of the associations of Rap1 with these proteins were independent of the TRF2 binding requirement [
      • Khattar E
      • Maung KZY
      • Chew CL
      • et al.
      Rap1 regulates hematopoietic stem cell survival and affects oncogenesis and response to chemotherapy.
      ]. These observations also suggest that Rap1 has a role in the general DNA damage response.

      Rap1 in modulation of telomeric DNA damage response

      Rap1 is recruited to telomeres via TRF2, and as TRF2 has been found to have an essential role in repressing ATM signaling and the NHEJ pathway, it was initially thought that Rap1 might also contribute to this activity. However various studies have found that Rap1 makes no contribution in repressing NHEJ at telomeres or inhibiting ATM kinase signaling. The first such evidence was provided by studies in mouse embryonic fibroblasts (MEFs) with TRF2 or Rap1 deletion (–/–). TRF2–/– MEFs exhibited accumulation of proteins such as 53BP1, MDC1, and γ-H2AX at chromosomal ends (referred to as telomere dysfunction-induced foci [TIF]), activation of ATM pathway, and telomeric fusions. However, Rap1–/– MEFs did not exhibit any such TIFs or activation of ATM kinase signaling [
      • Sfeir A
      • Kabir S
      • van Overbeek M
      • Celli GB
      • de Lange T
      Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.
      ]. Furthermore, when the TRF2 mutant (which cannot interact with Rap1) was introduced into Rap1–/– MEFs and TRF2–/– MEFs, no telomere fusions or ATM kinase activation was observed, suggesting that TRF2 alone was necessary and sufficient to repress the NHEJ pathway and ATM kinase activity at telomeres. This is further supported by the finding that Rap1–/– mice are viable and fertile and display no lethal telomere dysfunction.
      Martinez et al. [
      • Martinez P
      • Gomez-Lopez G
      • Pisano DG
      • Flores JM
      • Blasco MA
      A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance.
      ] reported a protective role for Rap1 on mouse telomeres in Terc–/– mice. This was an interesting observation as it revealed that simultaneous deletion of Terc (which causes telomere shortening) and Rap1 led to telomere deprotection as reflected by increased multiple telomere signals, T-SCEs, and end-to-end chromosome fusions. The authors suggested that in studies where loss of Rap1 did not reveal any telomere deprotection, the reason could be the absence of critically short telomeres. In agreement with this finding, recently Lototska et al. [
      • Lototska L
      • Yue JX
      • Li J
      • et al.
      Human RAP1 specifically protects telomeres of senescent cells from DNA damage.
      ] reported that human Rap1 specifically prevents DNA damage by inhibiting the NHEJ pathway at critically short telomeres such as those found in senescent cells and cancer cells. Mechanistically, they described that Rap1 prevents accumulation of 53BP1 at telomeres and does not let the telomeres fuse. However, the role of TRF2 in this function of Rap1 requires further clarification.
      Additionally, in vivo studies in Rap1–/– mice have suggested that Rap1 is critical for repressing HDR at telomeres [
      • Sfeir A
      • Kabir S
      • van Overbeek M
      • Celli GB
      • de Lange T
      Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.
      ]. HDR at telomeres can result in T-SCEs, which if unequal could result in telomeric loss and, thus, could be deleterious to cells. It has been reported that loss of Rap1 in the absence of Ku70/80 (because it represses HDR at telomeres as well as at the genomic DSBs) in mice resulted in HDR activation at telomeres, leading to telomere recombination, and almost 10% of chromosomal ends had T-SCEs in metaphase MEFs [
      • Sfeir A
      • Kabir S
      • van Overbeek M
      • Celli GB
      • de Lange T
      Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.
      ]. Molecularly it was observed that Rap1, along with TRF2, represses PARP1 and SLX4 association with telomeres, which is the major factor contributing to HDR at telomeres [
      • Rai R
      • Chen Y
      • Lei M
      • Chang S
      TRF2-RAP1 is required to protect telomeres from engaging in homologous recombination-mediated deletions and fusions.
      ]. Their accumulation at telomeres in the absence of Rap1 and TRF2 resulted in rapid telomere resection and generation of telomere-free chromosomal fusions. The Myb domain of Rap1 has been found to be important in this activity. A more detailed understanding of this molecular mechanism is particularly important because alternative lengthening of telomeres (which is observed in almost 15% of cancers) utilizes HDR for telomere maintenance, and it would be interesting to investigate how Rap1 activity is regulated in this situation [
      • Cesare AJ
      • Reddel RR
      Alternative lengthening of telomeres: models, mechanisms and implications.
      ].

      Rap1 in modulation of nontelomeric DNA damage response

      Interaction of Rap1 with various DNA damage response proteins in the absence of TRF2 (as discussed under Structure of the Rap1 protein) suggests that it might have a role in regulating the nontelomeric DNA damage response. Recently, our group reported a novel function of Rap1 in regulating the survival of hematopoietic stem cells (HSCs), as well as in affecting oncogenesis and chemotherapeutic response [
      • Khattar E
      • Maung KZY
      • Chew CL
      • et al.
      Rap1 regulates hematopoietic stem cell survival and affects oncogenesis and response to chemotherapy.
      ]. Rap1 was found to serve as an adaptor in the NHEJ, pathway and this activity did not require its association with TRF2, suggesting that it was nontelomeric.
      The hematopoietic system is essential to the formation of blood throughout life. Hematopoietic stem and early progenitor cells (HSPCs) divide to produce lineage-committed progenitors, which further differentiate into mature blood cells of various types [
      • Birbrair A
      • Frenette PS
      Niche heterogeneity in the bone marrow.
      ]. The highly proliferative nature of the hematopoietic system makes it susceptible to DNA damage signals, and therefore, the DNA damage response becomes critical in regulating the survival and function of hematopoietic cells. Exposure of the body to irradiation and DNA-damaging chemotherapeutic drugs has been implicated in the development of bone marrow failure, myelodysplastic syndrome, and therapy-induced leukemia [
      • Bunting SF
      • Nussenzweig A
      End-joining, translocations and cancer.
      ,
      • Ceccaldi R
      • Rondinelli B
      • D'Andrea AD
      Repair pathway choices and consequences at the double-strand break.
      ]. Irradiation and chemotherapeutic drugs generally result in DSBs that can be repaired via either the NHEJ or the HDR pathway [
      • Kakarougkas A
      • Jeggo PA
      DNA DSB repair pathway choice: an orchestrated handover mechanism.
      ]. These pathways are very different in terms of the regulation, outcome, and molecular mechanisms. The HDR pathway is homology dependent; thus, it is error free and is mostly active in the S phase of the cell cycle. The NHEJ pathway is not dependent on homology; thus, it is considered error prone and is active throughout the cell cycle. During steady state, HSPCs are mostly quiescent and thus depend on the NHEJ pathway for repair of any DNA damage that occurs while the downstream progenitors are mostly actively dividing, and they utilize the HDR pathway to repair any DNA damage encountered [
      • Biechonski S
      • Olender L
      • Zipin-Roitman A
      • et al.
      Attenuated DNA damage responses and increased apoptosis characterize human hematopoietic stem cells exposed to irradiation.
      ]. Consequently, HSPCs have been found to be more prone to exogenous DNA damage resulting in bone marrow failure and leukemia. Thus, an understanding of the underlying factors regulating the DNA damage response in HSPCs becomes important.
      In our study, we observed that under steady-state conditions, loss of Rap1 had no significant effect on hematopoietic cell populations in spleen or bone marrow in mice. However, on irradiation at lethal dose or treatment with the DNA-damaging drug 5-fluorouracil, survival of Rap1–/– mice was significantly reduced compared with that of wild-type mice. At a sublethal dose of irradiation, it was observed that HSPCs in Rap1–/– mice were not able to proliferate and repopulate, resulting in bone marrow failure when compared with the wild-type mice. HSPCs from Rap1–/– mice exhibited increased levels of the DNA damage marker γH2AX, which was persistent over 2 weeks, suggesting loss of Rap1 results in genomic instability. This further implied that because of loss of Rap1, HSPCs were unable to repair the DNA damage. Whether the same phenotype is observed in other stem cells in Rap1–/– mice requires further investigation. Furthermore, using cancer cell line-based studies, we observed that depletion of Rap1 led to increased DNA damage on treatment with chemotherapeutic drugs that caused DNA DSBs. The DNA damage-induced foci did not overlap with telomeric signal, suggesting a telomere-independent role for Rap1 in mediating the DNA damage response. We, however, were not able to detect the presence of Rap1 directly at DNA damage foci.
      As the NHEJ pathway is the major repair pathway in HSPCs, we investigated the role of Rap1 in the NHEJ pathway. Using a cell-based double-vector system [
      • Bennardo N
      • Cheng A
      • Huang N
      • Stark JM
      Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.
      ], we found that reducing Rap1 levels resulted in reduced NHEJ pathway efficiency in human cancer cells. As the NHEJ pathway is also physiologically relevant in B-cell class switching, we investigated the role of Rap1 in this process and observed that Rap1–/– B cells exhibited significantly reduced IgG1 class switching.
      Genome instability is one of the hallmarks of cancer, and NHEJ pathway proteins play an important role in maintaining genome stability [
      • Hoeijmakers JH
      Genome maintenance mechanisms for preventing cancer.
      ,
      • Burma S
      • Chen BP
      • Chen DJ
      Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.
      ]. One of the outcomes of NHEJ pathway activity is chromosomal translocations, which also are a characteristic feature of hematological malignancies and are also known as initiating events in the process of transformation [
      • Bunting SF
      • Nussenzweig A
      End-joining, translocations and cancer.
      ,
      • Ghezraoui H
      • Piganeau M
      • Renouf B
      • et al.
      Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.
      ]. Mutations in these proteins are known to result in increased susceptibility to cancer. For example, Ku70-deleted mice have a higher incidence of thymic lymphomas [
      • Gu Y
      • Seidl KJ
      • Rathbun GA
      • et al.
      Growth retardation and leaky SCID phenotype of Ku70-deficient mice.
      ]. Similarly, in mice with an SCID mutant background, deletion of DNA-PKs along with p53 resulted in a rapid onset of lymphomas and leukemias [
      • Guidos CJ
      • Williams CJ
      • Grandal I
      • Knowles G
      • Huang MT
      • Danska JS
      V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors.
      ]. Similar to this, we also observed that deletion of Rap1 resulted in increased susceptibility of mice to cancer when crossed with cancer mouse models such as Eµ-Myc-induced lymphomagenesis and PyMT-driven breast cancer. Interestingly, we also observed that loss of Rap1 and reduction in Rap1 levels sensitized the mouse and human cancer cells, respectively, to various chemotherapeutic agents. Similar observations in human cancer cells have been reported previously [
      • Zha Y
      • Gan P
      • Yao Q
      • Ran FM
      • Tan J
      Downregulation of Rap1 promotes 5-fluorouracil-induced apoptosis in hepatocellular carcinoma cell line HepG2.
      ]. Rap1 levels have been found to increase in response to DNA-damaging agents, supporting our finding on the role of Rap1 as an adaptor in DNA repair [
      • Kato M
      • Nakayama M
      • Agata M
      • Yoshida K
      Gene expression levels of human shelterin complex and shelterin-associated factors regulated by the topoisomerase II inhibitors doxorubicin and etoposide in human cultured cells.
      ]. Recently it was reported that depletion of Rap1 led to more DNA damage upon irradiation in colorectal cancer cells, and higher Rap1 levels are associated with a poor prognosis in colorectal cancers [
      • Anuja K
      • Kar M
      • Chowdhury AR
      • et al.
      Role of telomeric RAP1 in radiation sensitivity modulation and its interaction with CSC marker KLF4 in colorectal cancer.
      ]. It has also been reported that mutations in Rap1 are associated with familial melanoma [
      • Aoude LG
      • Pritchard AL
      • Robles-Espinoza CD
      • et al.
      Nonsense mutations in the shelterin complex genes ACD and TERF2IP in familial melanoma.
      ]. Mutations in Rap1 have also been found to be present in various other cancer types; however, their functional significance needs to be investigated. As the NHEJ pathway is important in hematological malignancies such as leukemia, investigating the functional significance of Rap1 in human leukemia might be insightful. It would also be interesting to investigate the susceptibility of Rap1–/– tumors to chemotherapeutic agents and how mutations in Rap1 affect its function and promote cancer.
      At the molecular level, the NHEJ pathway minimally requires seven core proteins for its activity [
      • Davis AJ
      • Chen DJ
      DNA double strand break repair via non-homologous end-joining.
      ]. DNA DSBs are recognized and bound by Ku70 and Ku86 proteins, which then recruit the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) followed by Artemis, which performs end processing. Lastly, X-ray cross-complementing 4 (XRCC4), XRCC4-like factor (XLF), and the DNA ligase IV (LIG IV) complex are recruited for ligation of DSBs. We observed that Rap1 serves as an adaptor in the NHEJ pathway by mediating the interaction between XRCC4/LIG IV and DNA-PKcs, which is followed by their reduced association with chromatin and, thus, impact on DNA repair. It would be interesting to further study the molecular details of this novel adaptor role of Rap1 in DNA repair. In vitro electron microscopy studies by Arat and Griffith [
      • Arat NO
      • Griffith JD
      Human Rap1 interacts directly with telomeric DNA and regulates TRF2 localization at the telomere.
      ] suggested that mammalian Rap1 can bind to DNA directly. They further observed that Rap1 associates directly with DNA in sequence-independent mode and has higher affinity for double-strand/single-strand DNA junctions. They proposed that this DNA binding activity of Rap1 might be via its BRCT domain and might be important for its extra-telomeric role. However, in vivo studies have not been able to find this direct DNA binding activity of Rap1 yet. Thus, further efforts are required to investigate this DNA binding activity in the light of our findings on its role in DNA repair at genomic regions. As Rap1 has also been found to regulate transcription at extratelomeric regions, it also might be interesting to investigate its role in regulating the DNA damage response via gene expression regulation [
      • Yeung F
      • Ramirez CM
      • Mateos-Gomez PA
      • et al.
      Nontelomeric role for Rap1 in regulating metabolism and protecting against obesity.
      ].
      Figure 2 summarizes role of Rap1 in the DNA damage response at telomeric and nontelomeric regions.
      Figure 2
      Figure 2Role of Rap1 in DNA damage pathways at telomeric and nontelomeric regions. At telomeric regions, Rap1 associates with TRF2 protein. TRF2 and Rap1 inhibit PARp1 and SLX4, respectively, thus repressing homology-directed repair (HDR). At nontelomeric regions, DNA damage results in the formation of double-strand breaks (DSBs). This activates the nonhomologous end joining pathway (NHEJ), where Rap1 acts as an adaptor promoting association of XRCC4/ligase IV with DNA-PK, thus increasing its efficiency.

      Conclusions and future directions

      Mammalian Rap1 has been investigated mostly in the context of the shelterin component with function at telomeres. However, in the past decade, several studies have revealed its role as an adaptor in functional complexes formed under various physiological conditions. Its role in the DNA damage response at nongenomic regions is particularly important and interesting. DNA damage also controls stem cell survival, and Rap1 expression under those conditions becomes particularly important in maintaining their homeostasis. The molecular mechanisms underlying Rap1 regulation of various aspects of the DNA damage response and regulation of stem cell survival after induction of DNA damage merit further investigation. Additionally, the functional significance of genetic mutations in Rap1 in humans and how it leads to cancer susceptibility should be explored.

      Acknowledgments

      EK is supported by research grants from the Department of Biotechnology (No. BT/RLF/Re-entry/06/2015 ) and Department of Science and Technology (No. ECR/2018/002117 ) and by an NMIMS Seed Grant (No. IO 401405 ).

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