The Role of the MRN Complex in Cancer – F Zeilstra

The Role of the MRN Complex in Cancer

F Zeilstra. University Medical Center Groningen, Groningen, The Netherlands. July 12th 2010.

Exogenous or endogenous DNA damaging agents can threaten the genomic integrity of a cell. Therefore, there are cell cycle checkpoints to prevent passing on genomic instability as well as DNA repair mechanisms. Two major double-strand break repair mechanisms exist; non-homologous end joining (NHEJ) and (HR). The MRN complex is involved in both processes and especially plays a pivotal role in HR together with ataxia telangiectasia mutated (ATM). Mutations in the complex or the ATM gene cause severe disorders, sensitivity to radiation and predisposition to several types of cancer because of the failure to repair DSBs. However, the protein complex also appears to be an attractive target for anticancer therapies due to its involvement in both repair machinery.

Keywords: Nijmegen breakage syndrome, NBS, NBN, , MRE11, ATM, ATLD.

Introduction simply used as a template to fix the break, while DSBs are more complex and can All organisms have several mechanisms to cause rearrangements in the maintain the integrity of their genome for chromosomes, cell death, cell senescence generations. These include cell cycle or tumors if not properly repaired [Khanna checkpoints during DNA replication and and Jackson, 2001; Van Gent et al, 2001; mitosis as well as DNA damage repair. Helmink et al, 2009; Shiloh 2003; Rouse The latter is important because genomes and Jackson, 2002; Wyman and Kanaar, are subjected to various types of 2006]. exogenous or endogenous DNA damaging agents, like radiation or reactive radicals, There are two major DSB repair respectively [Khanna and Jackson, 2001; mechanisms: non-homologous end joining Shen and Nickoloff, 2007; Paques and (NHEJ) and homologous recombination Haber, 1999; Franco et al, 2006; (HR) with very distinct differences and little Chaudhuri et al, 2007; Murnane, 2006; overlap (Fig. 1) [Czornak et al, 2008; Acilan et al, 2007; Ward, 1998; Limoli et Helmink et al, 2009]. Non-homologous end al, 2002; Bosco et al, 2004]. These agents joining is mostly used when the DNA have several damaging effects, such as DSBs are caused before the replication in cell death or genetic alterations, including the G0/G1-phase of the cell cycle and mutations, deletions, translocations, simply re-ligates broken DNA ends. chromosome loss and DNA breaks However, this can cause this process to [Alberts et al, 2002]. The consequences of be imprecise and error prone [Wyman and DNA breaks can lead to genome instability Kanaar, 2006]. Unlike NHEJ, homologous and , amongst others [Su, recombination repairs DSBs before the 2006; Czornak et al, 2008]. cell enters mitosis. It occurs during and shortly after DNA replication, in the S- and There are two types of DNA breaks; G2-phase. The sister chromatid is used as single-strand breaks (SSBs) and double- a template for a precise repair [Wyman strand breaks (DSBs) [Watson et al, 2004]. and Kanaar, 2006; Alberts et al, 2008; In case of the SSBs the other strand is The Role of the MRN Complex in Cancer – F Zeilstra

Saleh-Gohari and Helleday, 2004; Sonoda Non-homologous end joining (NHEJ) et al, 2006]. Double-strand breaks in multi-cellular The repair mechanisms both use several eukaryotes can be repaired via non- proteins and kinases, which are of great homologous end joining. While this can importance for the proper repair of DSBs. occur throughout the entire cell cycle, in One of the protein complexes that seems most cases they only revert to NHEJ to be involved in both processes is known during the G0/G1-phase [Lieber, 2008; as the MRE11/RAD50/NBS (MRN) Wyman and Kanaar, 2006]. complex. It especially seems to play a pivotal role in HR. Unfortunately, recent NHEJ is considered to be distinctively studies have shown that this complex is flexible because of the nuclease, affected in several types of cancer. Even polymerase and ligase activities used. when one of the genes of the MRN This flexibility allows NHEJ to function on a complex is unable to provide a proper wide range of configurations that are the working protein it can have dire result of DSBs. Especially when these consequences for an individual. breaks are caused by oxidative damage or ionizing radiation [Lieber, 2008]. This thesis will elaborate on the function of the MRN complex and its involvement in A homologous chromosome is not the repair of DNA double-strand breaks. In needed because the severed DNA ends addition, the role of the complex in various are rejoined by NHEJ [Sonoda et al, 2006; types of cancer will be discussed. Moore and Haber, 1996]. This allows loss Furthermore, it will be examined whether of nucleotides or even addition of there could be a role for the MRN complex complementary bases (micro-homology) at in the treatment or targeting of cancer. the rejoining site [Lieber, 2008; Shrivastav et al, 2008]. That is why this process is considered error prone and imprecise [Wyman and Kanaar, 2006; Lieber, 2008].

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The Role of the MRN Complex in Cancer – F Zeilstra

However, if there are DSBs with due to DSBs in developing G1-phase complementary overhangs and lymphocytes, which is very similar to ATM- 5’phosphates and 3’hydroxyl groups, deficient lymphocytes [Helmink et al, 2009]. these can be precisely repaired by NHEJ

[Clikeman et al, 2001; Lin et al, 1999]. An important protein kinase that is activated by DNA DSBs is ataxia The repair of DSBs through NHEJ requires telangiectasia mutated (ATM), encoded by three enzymatic activities; nucleases to the ATM gene. This protein remove damaged DNA, polymerases to phosphorylates several other proteins that aid in the repair and a ligase to restore the function in DNA repair and cell cycle backbone (Fig. 2) [Lieber, 2008]. In checkpoints when there is DNA damage mammalian cells the [Boultwood, 2001; Taylor and Byrd, 2005]. MRE11/RAD50/NBS1 (MRN) complex is When ATM is lacking in cells, DNA-PK can also involved, preceding the binding of Ku phosphorylate histone H2AX, termed γ- [Shravastay et al, 2008] but it does not H2AX [Burma et al, 2001; Collis et al, seem to be required for NHEJ [Rodrigue et 2005; Cui et al, 2005; Shao et al, 1999; al, 2006; Yang et al, 2006]. This raises the Burma and Chen, 2004; Chan et al, 1999; question of what the exact role of the Karmaker et al, 2002; Yannone et al, complex is if it is not required but involved 2001; Stucki and Jackson, 2006]. Since when present. Especially considering the NHEJ simply ligates DNA ends without the NHEJ repair activity was not affected in use of a homologue chromosome to fix the Xenopus laevis by the addition of MRN DSBs, the local DNA does not return to its [Huang and Dynan, 2002; Di Virgilio and original sequence which explains the wide Gautier, 2005] even though a deficient range of end products after NHEJ MRN complex after V(D)J recombination [Shravastay et al, 2008]. does lead to more unrepaired coding ends

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The Role of the MRN Complex in Cancer – F Zeilstra

Homologous recombination (HR) al, 2008; Brugmans et al, 2007]. However, there are indications from studies The genomic stability of somatic cells is observing HR for DSBs in yeast that point maintained through precise repair of mutations adjacent to the site of repair are double-stranded DNA breaks induced by more frequent [Strathern et al, 1995]. exogenous and endogenous agents, such Another problem for HR is that, with the as damaged replication forks and repair of exception of sister chromatids, the incomplete using a mechanism templates used for the repair are often not called homologous recombination (HR) entirely homologous. This can result into [San Filippo et al, 2008]. HR only occurs loss of heterozygosity due to gene during the S-phase and G2-phase of the conversion [Nickoloff, 2002]. cell cycle, before mitosis takes place in The process of homologous recombination order to prevent the passing on to in order to repair DSBs involves several daughter cells and thereby preserving the steps (Fig. 3), namely: end resection, genomic integrity [Alberts et al, 2008]. strand invasion, synthesis, ligation, branch Homologous recombination is considered migration and holiday junction (HJ) to be more precise than NHEJ because of resolution [Sharan and Kuznetsov, 2007; the usage of homologous chromosomes, San Filippo et al, sister chromatids or repeated regions in 2008]. In the next paragraph these steps the genome in order to restore the double- will be discusses in more detail. stranded breaks and can be up to one hundred percent accurate [Shrivastav et

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The Role of the MRN Complex in Cancer – F Zeilstra

Step 1 – end resection [Shrivastav et al, 2008; Czornak et al, After the DSB has been formed, 2008; Sharan and Kuzentsov, 2007; San homologous recombination is initiated with Filippo et al, 2008; Liu and West, 2004; end-processing at the broken ends [San Holliday, 1964; Mizuuchi et al, 1982]. Filippo et al, 2008], regulated by the MRN complex (the yeast homolog is called A number of homologous recombination MRE11/RAD50/XRS2 – MRX – complex) factors involved in HR include RAD51, the and another exonuclease [Krogh and MRE11/RAD50/NBS1 (MRN) complex, Symington, 2004; Shrivastav et al, 2008; BRCA2, RPA, Dmc1, PALB2, DSS1, San Filippo et al, 2008] This results in 3’ RAD52, and RAD54 (Table 1) [San Filippo single-stranded DNA (ssDNA) tails, which et al, 2008; Shrivastav et al 2008; will be used as templates for finding the Czornak et al, 2008]. There appears to be homologous DNA sequence in the sister a role for BRCA1 as it interacts with chromatid. BRCA2 and several other proteins that are considered repair factors, but as of yet the Step 2 – strand invasion exact role is unclear in HR as well as Once the first step has been completed, NHEJ [Huang and D’Andrea, 2006]. the ssDNA tails are used to invade the sister chromatid. A recombinase filament In addition, homologous recombination is formed on the ssDNA ends, after which plays an important role in meiosis by the homologous DNA is invaded to form a mediating an exchange of genetic D-loop. information between the paternal and maternal alleles within the gamete Step 3 – DNA synthesis precursor cells, thereby ensuring genetic DNA polymerases that have yet to be diversity among the offspring of common identified [Maolisel et al, 2008] extend the parent and ensures proper segregation at 3’ end of the invading strand after allowing the first meiotic division via cross over base-pairing of the invading and the [Neale and Keeny, 2006; San Filippo et al, complementary strands. 2008].

Step 4 – DNA ligation The second DSB end is annealed to the MRE11/RAD50/NBS (MRN) complex extended D loop and the two crossed strands form Holliday junctions (HJs) [Holliday, 1964]. In order to repair the damaged DNA, several kinases such as ataxia- Step 5 – branch migration telangiectasia mutated (ATM), ataxia- Holiday junctions can branch migrate telangiectasia and Rad-3-related (ATR) either way; by returning in the direction of and DNA protein kinase catalytic subunit the replication site it can reverse the invasion, leaving an extension beyond the (DNA PKcs) phosphorylate and thereby DSB. The resulting ssDNA end can be activate several protein targets. Upon repaired by SSA or NHEJ. Or the HJ can activation, these kinases activate DNA proceed in the opposite direction and repair. If the damage is irreparable these invade the same homologous DNA kinases direct the cell to programmed cell molecule forming a double HJ structure. death, apoptosis [Shiloh, 2003a; Abraham,

2004]. Step 6 – Holiday junction resolution The HJs are resolved, which leads to crossover or non-crossover products, depending on the cleavage site and the remaining gaps ad nicks of ssDNA are repaired by DNA polymerase and ligase The Role of the MRN Complex in Cancer – F Zeilstra

One protein complex that plays a pivotal As previously described, ATM role in this signaling cascade is called the serine/threonine kinase is an important MRN complex. Together with these initiator of DNA damage response. It is checkpoint kinases, the MRN complex is recruited and activated by MRN when involved in genomic stability as well as there are DNA DSBs [Maser et al, 1997; maintenance, and mutations in Nelms et al, 1998; Difilippantonio et al, the genes encoding for these proteins lead 2005; Uziel et al, 2003; Lee and Paull, to severe disorders that affect the repair 2004; Lee and Paull, 2005]. However, mechanism [Czornak et al, 2008; Jazayeri there are indications that ATM might be et al, 2008].The MRN protein complex activated independent of the MRN consists of the MRE11, RAD50 and NBS1 complex, for example when DSBs are proteins and is an important element in generated at stalled replication forks DNA replication, repair and signaling to during S-phase [Burma et al, 2001; Collis the cell cycle checkpoints. As mentioned et al, 2005; Cui et al, 2005; Shao et al, in the previous sections, the MRN complex 1999; Burma and Chen, 2004; Chan et al, is involved in the early steps of 1999; Karmaker et al, 2002; Yannone et homologous recombination and it appears al, 2001; Stucki and Jackson, 2006; to play a role during non-homologous end Difilippantonio et al, 2005; Helmink et al, joining as well. While it is involved in both 2009; Paulsen and Cimprich, 2007]. In this major reparation mechanisms [D’Amours case, the DSBs occur naturally when the and Jackson, 2002; Van den Bosch et al, replication forks encounter blocking 2003], it primarily functions in cells with lesions deriving from metabolic DNA double-strand breaks that have byproducts, e.g. reactive oxygen species already undergone replication and thus will (ROS) and this leads to fork collapse (Fig. be repaired by homologous recombination 4) [Shen and Nickoloff, 2007]. Since this [Stracker et al, 2004; D’Amours and produces only a one-ended double-strand Jackson, 2002]. break, also known as double-strand end (DSE) it is assumed fork collapses are

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The Role of the MRN Complex in Cancer – F Zeilstra repaired only by HR and not NHEJ 2001; Wiltzius et al, 2005; Hopfner et al, [Shrivastav et al, 2008]. This suggests that 2002; Moreno-Herrero et al, 2005]. the requirement for the MRN complex to initiate a response to double-strand breaks NSB1 is context-dependent. The last protein of the MRN complex is NBS1 and stimulates endonuclease MRE11 activity [Paull and Gellert, 1999; Trujillo The MRN complex can bind to the broken and Sung, 2001]. It is composed of three ends of double-stranded DNA breaks due regions; the N-terminus, a central region to MRE11 and RAD50 [Maser et al, 1997; and the C-terminus. The N-terminal region De Jager et al, 2001b; Hopfner et al, contains a fork-head associated (FHA) 2002]. This association between RAD50 doman and two breast cancer C-terminus and MRE11 during the DNA processing (BRCT) domains (BRCT1 and BRCT2) stimulates the exonuclease and [Bork et al, 1997; Callebaut and Mornon, endonuclease activities of MRE11 1997; Becker et al, 2006]. ATM or ATR [Moreno-Herrero et al, 2005; Paull and kinases are able to phosphorylate NBS1 Gellert, 1999; Trujillo and Sung, 2001] and due to the central region which is forming dimers it can bridge two DNA ends composed of several consensus during HR-mediated double-strand breaks sequences [Lim et al, 2000; Zhao et al, repair [Stracker et al, 2004; D’Amours and 2000; Wu et al, 2000]. The C-terminus Jackson, 2002; Williams et al, 2008]. contains a MRE11 binding domain [Desai-

RAD50 Mehta et al, 2001] and ATM recruitment The RAD50 component of the MRN motif [Falck et al, 2005]. complex is a DNA binding protein. This binding property of RAD50 is possibly ATM phosphorylation activates the MRN involved in tethering sister chromatids complex during homologous recombination Proteins of the MRN complex are [Anderson et al, 2001; De Jager M et al, phosphorylated by ATM in response to

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The Role of the MRN Complex in Cancer – F Zeilstra double-stranded DNA breaks and this Stracker et al, 2007; Difilippantonio et al, could modulate their functions in DSBs 2005; Cerasoletti and Concannon, 2004; responses [Matsuoka et al, 2007; Gatei et Falck et al, 2005; Cerasoletti et al, 2006; al, 2000; Wu et al, 2000; Lim et al, 2000]. Digweed et al, 1999; Laving et al, 2007]. Moreover, Rad50, NBS1 and MRE11 each have their own properties and together The fact that recruitment of ATM takes they function as a holocomplex with an place in short time suggests that it might important role in the repair of double- be partly activated before localization of stranded DNA breaks. The entire complex DSBs [Bakkenist and Kastan, 2003]. is also capable of partly unwinding and However, the partially activated ATM dissociating the 3’ overhang of the DNA cannot phosphorylate MRN-dependent duplex, which is probably one of the substrates such as BRCA1 [Kitagawa et reasons the MRN complex is successful in al, 2004]. It is possible that CtlP, a cell processing double-stranded breaks repair cycle regulation protein provides the MRN [Paul and Gellert, 1999]. complex and BRCA1 with a physical connection [Limbo et al, 2007]. The MRN complex and ATM are interdependent Sensing versus repair The initial recognition of and response to It is thought that the MRN complex and DNA double-strand breaks is the binding replication protein A (RPA) both sense of the MRN complex, by binding the DNA damage and activate a DNA damage broken ends together in preparation of response in the form of the ATM and ATR repair. This requires a swift response from proteins [Lee and Paull, 2004; Robison et the protein complex and the fact that ATM al, 2007]. While RPA is the major single- is activated just as rapid makes it difficult strand DNA (ssDNA) binding protein and to discern the exact sequence of events essential for replication, DNA repair and [Bakkenist and Katan, 2003; Kozlov et al, recombination [Zou et al, 2006], MRE11 2003; Lavin et al, 2007]. It was shown that protein binds ssDNA as well as double- the MRN complex is upstream of ATM strand DNA (dsDNA) [De Jager et al, activation, which suggests that it is indeed 2001a]. The 3’-ssDNA is a binding site for the MRN complex that can sense the DNA RPA and is processed by the nuclease damage [Lee and Paull, 2004]. Depleting activity of MRE11 [White and Haber, NBS and ATLD cells of the MRN complex 1990]. Further nuclease activity is inhibited during viral infection showed that ATM by the assembly of the RPA-ssDNA activation was retarded and reconstitution complex, which also provides the site for of the complex restored the ATM response repair [Sugiyama et al, 1997; Williams et [Uziel et al, 2003]. al, 2007].

However, Difilippantonio et al showed The MRN complex binds to the DNA without involvement of the ATM kinase as different results in mouse mutants, which it senses DSBs and promptly activates suggested that the ATM activation was not ATM [Mirzoeva and Petrini, 2003; Lavin, as severely reduced when the C-terminus 2007]. After activation there is a rapid was affected [Difilippantonio et al, 2007]. response from ATM. It phosphorylates Moreover, several other studies had several protein targets, activates signaling similar conflicting results and thus it is cascades and induces cell cycle arrest believed that the MRN complex and ATM [Dupre et al, 2006; Bakkenist and Kastan, 2003; Kozlov et al, 2006; Berkovich et al, are interdependent when recognizing and 2007; Canman et al, 1998; Khanna et al, signaling DSBs [Lee and Paull, 2004; 1998;Waterman et al, 1998] or cAbl

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The Role of the MRN Complex in Cancer – F Zeilstra

[Baskaran et al, 2001; Shafman et al, as recessive and is caused by mutations 1997]. in the ATM gene and leads to, amongst others, radio sensitivity, chromosomal There are two hypothesizes regarding translocations, immunodeficiency and sensing of DNA damage and subsequent repair; the first suggests that damage is cancer predisposition because of total loss sensed after which there will be repair, of ATM [Savitsky et al, 1995; Mavrou et al, while the second does not discriminate 2008]. In vivo studies have shown that between sensing and repair. Considering ATM is an inactive dimer that dissociates that both ATM and the MRN complex are into active monomers once double-strand involved in DNA damage repair machinery breaks are induced by ionizing radiation and work interdependent of each other, it [Bakkenist and Kastan, 2003; Shiloh, seems more plausible that indeed it is one and the same instead of two different 2006]. processes. Mutations in meiotic recombination 11 (MRE11) causing ataxia telanciectasia-like Cancer and other disorders disorder (ATLD) Mutations in MRE11 is responsible for A proteomic analysis has shown that there ATLD and bi-allelic mutations are over 700 proteins involved in the DNA extremely rare [Stewart et al, 1999; Delia repair mechanism and all of them were et al, 2004; Fernet et al, 2005]. Patients phosphorylated by ATR kinases and ATM with this disorder are very sensitive to in response to DNA damage [Matsuoka et radiation and rearrangements involving al, 2007]. Substrates of both ATM and chromosomes 7 and 14, but at a lower ATR kinases influence and engage in cell rate than in A-T [Stewart et al, 1999]. cycle regulation, metabolism, structure and Considering the rarity of this disorder not a proliferation as well as signal transduction, lot of research has been done. The studies immunity and oncogenesis and therefore it that have been performed only have a is suffice to say that dysfunction of either minimum of patients because there are kinase can lead to serious disorders simply not a lot of people with this leaving one vulnerable to DNA damaging disorder. That is also the reason why there agents and DSBs. The MRN complex is a lack of knowledge of the obviously plays a pivotal role in repairing consequences of ATLD, because so far double-stranded DNA breaks, whether that there are no immune deficiencies or is through NHEJ or HR, and this is malignancies reported for those patients supported by the fact that dysfunction of [Taylor et al, 2004]. However, like A-T, particular proteins involved in these they do suffer from hypersensitivity to mechanisms lead to severe disorders. ionizing radiation and genomic instability Ataxia telangiectasia (A-T), A-T-like [Hernandez et al, 1993; Klein et al, 1996; disorder (ATLD), Nijmegen breakage Stewart et al, 1999]. syndrome (NBS) and NBS-variant are prime examples of such disorders. In the Mutations in (NBN) gene causing next few paragraphs these disorders and Nijmegen breakage syndrome (NBS) their characteristics as well as several The gene that encodes for the NBS1 other genes involved in DNA repair protein, NBN, is possibly a tumor mechanisms will be discussed. suppressor gene due to the effects that people with Nijmegen breakage syndrome Mutations in Ataxia-telangiectasia mutated (NBS) suffer but this has yet to be proven (ATM) causing ataxia telangiectasia (A-T) [Dzikiewicz-Krawczyk, 2008]. However, A-T is an autosomal disorder, rare as well

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The Role of the MRN Complex in Cancer – F Zeilstra people with NBS do have problems with without the immunodeficiency [Gennery, their DNA repair mechanisms because 2006]. their MRN complex does not function properly, leaving them prone to DSBs Mutations in the MRN complex [INBSSG, 2000; Digweed and Sperling, As stated before, the MRN complex plays 2004].NBS is rare, caused by mutations in a pivotal role in the DNA repair machinery. the NBN gene and is also an autosomal It is thought to be of vital importance to recessive chromosome instability disorder maintain cell integrity considering null [Carney et al, 1998; Varon et al, 1998]. It mutations in one of the three components is characterized by microcephaly, of the MRN complex lead to embryonic immunodeficiency, growth retardation and lethality in mice [Xiao and Weaver, 1997; a very high cancer incidence, probably Luo et al, 1999; Zhu et al, 2001]. The due to its involvement in cell cycle result of mutations in this vital complex checkpoints and DNA damage response cause an instable genome due to lacking proteins [Becker et al, 2006]. A number of of proper working DSBs repair malignancies, like melanoma [Debniak et mechanisms and mutations in the ATR al, 2003; Steffen et al, 2006], non-Hodgkin genes leads to Seckel syndrome in lymphoma [Steffen et al, 2004; humans [O’Driscoll et al, 2003]. In Chrzanowska et al, 2006; Steffen et al, addition, there are other syndromes 2006], acute lymphoblastic leukemia associated with defects of other members [Varon et al, 2000; Resnick et al, 2003; in the DNA damage repair machinery, Chrzanowska et al, 2006], breast cancer such as Artemis deficiency [Moshouse et [Gorski et al, 2005; Kanka et al, 2007; al, 2001], Bloom syndrome (BS), Werner Sokolenko et al, 2007; Bogdanova et al, syndrome (WS) [Taniguchi and D’Andrea, 2008], and prostate cancer [Cybulski et al, 2006] and DNA ligase IV deficiency (LigIV) 2004; Hebbring et al, 2006], have been [O’Driscoll et al, 2001]. observed more frequently in heterozygote Mutations in breast cancer type 1 carriers of the NBN founder mutation. susceptibility protein (BRCA1) gene There is also overlap between NBS, A-T BRCA1 is a breast cancer tumor and ATLD [INBSSG, 2000; Digweed and suppressor protein and has a role in NHEJ Sperling, 2004] and it is estimated that that possibly involves chromatin about 200 people worldwide suffer from remodeling through the this disorder [Varon et al, 1998]. The ubiquitylation pathway [Huang and mutation leads to two proteins, NBNp26 D’Andrea, 2006] or MRE11 modulation and NBNp70 and while the shorter [Durant and Nickoloff, 2005]. There is an fragment, NBNp26 does not seem to be alternative Ligase III-mediated NHEJ associated with the MRE11 complex, the pathway involving PARP-1 but this is even longer fragment is associated with it more error-prone than the classical NHEJ [Maser et al, 2001]. [Wang et al, 2006]. Mutations in RAD50 causing Nijmegen Mutations in the breast cancer type 2 breakage syndrome-variant (NBS-variant) susceptibility protein (BRCA2) gene The NBS-variant is caused by mutations in Patients with mutations in the BRCA2 the RAD50 gene [Gennery, 2006] and so gene are predisposed to breast, ovarian far only one patient with two germline and other types of cancer [Jasin, 2002]. A mutations has been reported [Bendix- cancer-associated truncation or BRCA2 Waltes et al, 2005]. The symptoms of the impairs the transport of RAD51, an disorder are very similar to NBS but important protein involved in HR, to the

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The Role of the MRN Complex in Cancer – F Zeilstra nucleus [Davies et al, 2001]. Furthermore, affected response to, impairments or it has been shown that BRCA2 binds DNA failure of DNA repair regarding double- [Yang et al, 2005; San Filippo et al, 2006; strand breaks, treatment of cancer Yang et al, 2002; Martin et al, 2005], is patients with an affected MRN complex is needed for DNA damage-induced nuclear difficult at best. Individuals with improper RAD51 foci [Jasin, 2002; Yuan et al, 1999] working DNA repair machinery or any type and physically interacts with RAD 51 of DNA damage are highly sensitive to [Jasin, 2002; Sharan et al, 1997; Chen et radiation and thus regular chemo- or al, 1998; Wong et al, 1997]. radiotherapy does not suffice because these treatments are based on damaging Mutations in other genes involved in DNA the DNA of malignant tumors [Harper and repair Elledge, 2007]. Moreover, these people It is known that there are many genes, should not even be considered for these proteins and kinases involved in the DNA types of therapy. damage repair machineries. Therefore, it should come as no surprise that a lot of Normally, once the MRN complex functions of these components are still activates ATM or ATR a signaling cascade unknown. Small mutations in a single gene is set in motion to halt the cell cycle. can have dire consequences for an Checkpoint proteins 1 and 2 are key individual and at the same time these downstream substrates of ATM and ATR mutations can be so rare that they and (Fig. 5) [Gatei et al, 2003; Matsuoka et al, their subsequent disorders go unnoticed. 2000]. However, if the MRN complex has a Above mentioned disorders are known dysfunction in one of its components, be it best because of the consequences that MRE11, RAD50 or NBS there will be no lead to severe limitations of repair cell cycle arrest unless there is a different machinery, immune response, pathway to obtain the same result. In neurological and growth development and addition, activation of ATM also hypersensitivity to radiation and genomic contributes to the phosphorylation of , instability. However, considering the a tumor suppressor protein and this collaboration between all kinds of proteins indirectly trans-activates the kinase there is a good chance there are even inhibitor p21 [Matsuoka et al, 2007]. more disorders that are yet to be discovered. Of course, in view of the Different treatment for affected MRN importance of DSBs repair chances are complex that, like in mice, some of these disorders Other means of treatment are needed for in humans lead to embryonic lethality, individuals with a defective MRN complex. which can hinder further understanding of It is possible that there are more pathways DNA repair machinery. to repair DNA damage than is currently known, considering a lot of patients do not respond to the normal cancer treatments as well as expected. However, such The MRN complex and cancer pathways need to be activated in order to treatment work. If the MRN complex will not work The MRE11/RAD50/NBS (MRN) complex properly perhaps it can be sidestepped or activated otherwise. plays a pivotal role in the sensing of DNA damage as well as activating the repair mechanisms. Considering that mutations in the MRN complex leads to a severely

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The Role of the MRN Complex in Cancer – F Zeilstra

Non-homologous end joining as well as Several studies have already proven that homologous recombination uses the MRN disrupting the organized structure of the complex to repair double-strand breaks. MRN complex effectively disrupts the Conversely, there are several other repairment of double-strand DNA breaks pathways to repair DNA damage, such as [Stewart et al, 1999; Girard et al, 2000; direct reversal of DNA damage Cariveau et al, 2007; Dupre et al, 2008]. [Sedgewick et al, 2007], MMR pathway Not to mention the effects the earlier [Jiricny, 2006; Vasen et al, 2007], NER mentioned disorders involving the MRN pathay [Truglio et al, 2006], complex have on the repair machinery.

BER pathway [Wilson and Bohr, 2007; Cheadle and Sampson, 2007].

The MRN complex as a target For other types of cancer, in individuals who do not suffer from an affected MRN complex the protein complex is an attractive target for anticancer treatments.

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The Role of the MRN Complex in Cancer – F Zeilstra

Conclusion malignancies as there is a clear association between DNA damage and Double-strand DNA breaks (DSBs) are cancer. dangerous if not properly repaired. Thankfully, there are several repair The exact mechanism of DNA sensing is mechanisms, the two major ones being still unclear, even if there are several non-homologous end joining (NHEJ) and hypotheses. In addition, the precise role of homologous recombination (HR). Both the MRN complex in NHEJ still eludes us machineries have their advantages but as well as how the choice between NHEJ NHEJ seems to be more error-prone than and HR is regulated, considering NHEJ HR under most circumstances. However, can occur at any time during the cell cycle. different circumstances call for different It is also still unknown what happens to the approaches as well as different MRN complex after the DSBs are repaired. mechanisms. The MRE11/RAD50/NBS1 (MRN) complex is involved in both NHEJ The protein complex appears to be an and HR and obviously plays a pivotal role attractive target for anticancer treatments in the repair of double-strand DNA breaks because of the signaling cascade it and when affected the consequences are initiates after sensing DNA damage. grave. However, considering the importance of the role of the MRN complex further The protein complex senses DNA damage research is needed to make sure it is a and initiates the repair mechanisms in suitable target. Particularly when order to maintain genomic integrity. There examining the results a malfunctioning are several disorders with a clear complex has in A-T, ATLD, NBS or NBS- predisposition for cancer that are the direct variant patients. effect of a malfunction in the MRN complex. Only one of the components has It is clear that patients with an affected to be affected to ensure that an individual MRN complex or ATM gene require is unable to properly use homologous different treatment than regular cancer recombination or even non-homologous patients considering their sensitivity to end joining to repair double-strand breaks radiation, lack of DNA repair machinery in the DNA. A direct result of this is that and immune deficiencies. Especially, these patients are highly sensitive to considering that chemo- and radiotherapy radiation, not to mention that they often are based on causing DNA damage. suffer from neurological defects and Conversely, there are no cures or proper immune deficiencies as well. treatments for disorders like these to date.

In combination with ATM the MRN While there have been numerous studies complex and their transducer and to examine the precise effect of the mediator proteins forms an efficient presence of the MRN complex, there are network that senses and signals DNA still many unanswered questions. The damage and activates the proper repair research into this particular subject is mechanisms. Considering this highly hindered by ethical situations as well as branched network depends on several lack of patient material, due to the proteins and kinases in order to repair rareness of some of the disorders DSBs, a defective key role component can mentioned earlier. In addition the question have disastrous consequences, such as arises if in vitro studies can be translated instable genome disorders as well as to the human body considering the making one prone to various types of differences between cultures or germ lines

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The Role of the MRN Complex in Cancer – F Zeilstra and the protein interactions in a living In summary, further research is needed to person. enhance our comprehension of the MRN complex, DSBs repair machineries and Furthermore, many studies are focusing their relation to cancer. Moreover, if it is on mouse, frog or yeast models while possible to unravel the mysteries involving there is no guarantee that it is the same in DNA double-strand repairs as well as the humans. Although several human exact role of the MRN complex it can save homologous have been found there are the lives of millions of cancer patients still many unknown. worldwide.

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The Role of the MRN Complex in Cancer – F Zeilstra

Acknowledgements

 Marcel van Vugt for supervising the progress of this thesis as well as providing useful feedback.

References

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