The Role Of The Mrn Complex In Cancer Biology Essay


All organisms have several mechanisms to maintain the integrity of their genome for generations. These include cell cycle checkpoints during DNA replication and mitosis as well as DNA damage repair. The latter is important because genomes are subjected to various types of exogenous or endogenous DNA damaging agents, like radiation or reactive radicals, respectively [Khanna and Jackson, 2001; Shen and Nickoloff, 2007; Paques and Haber, 1999; Franco et al, 2006; Chaudhuri et al, 2007; Murnane, 2006; Acilan et al, 2007; Ward, 1998; Limoli et al, 2002; Bosco et al, 2004] leading and the consequences of DNA breaks can lead to genome instability and carcinogenesis, amongst others [Su, 2006; Czornak et al, 2008].

There are two types of DNA breaks; single-strand breaks (SSBs) and double-strand breaks (DSBs) [Watson et al, 2004]. In case of the first the other strand is simply used as a template to fix the break, while the latter are more complex and can cause rearrangements in the chromosomes, cell senescence, tumors or cell death if not properly repaired [Khanna and Jackson, 2001; Van Gent et al, 2001; Helmink et al, 2009; Shiloh 2003; Rouse and Jackson, 2002; Wyman and Kanaar, 2006].

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There are two major DSB repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR) with very distinct differences and little overlap (Fig. 1) [Czornak et al, 2008; Helmink et al, 2009]. Non-homologous end joining is mostly used when the DNA DSBs are caused before the replication in the G0/G1-phase of the cell cycle and simply re-ligates broken DNA ends, which can cause this process to be imprecise and error prone [Wyman and Kanaar, 2006]. Homologous recombination repairs DSBs before mitosis; it occurs during and shortly after DNA replication, in the S- and G2-phase and uses the sister chromatid as template for a precise repair [Wyman and Kanaar, 2006; Alberts et al, 2008; Saleh-Gohari and Helleday, 2004; Sonoda et al, 2006].

Non-homologous end joining (NHEJ)

Double-strand breaks in multi-cellular eukaryotes can be repaired via non-homologous end joining and this can occur throughout the entire cell cycle, but in most cases they only revert to NHEJ during the G0/G1-phase [Lieber, 2008; Wyman and Kanaar, 2006].

NHEJ is considered to be distinctively flexible because of the nuclease, polymerase and ligase activities used and this flexibility allows NHEJ to function on a wide range of configurations that are the result of DSBs, especially when these breaks are caused by oxidative damage or ionizing radiation [Lieber, 2008].

A homologous chromosome is not needed [Sonoda et al, 2006; Moore and Haber, 1996] because the severed DNA ends are rejoined by NHEJ and this allows loss of nucleotides or even addition of complementary bases (micro-homology) at the rejoining site [Lieber, 2008; Shrivastay et al, 2008], which is why this process is considered error prone and imprecise [Wyman and Kanaar, 2006; Lieber, 2008]. However, if there are DSBs with complementary overhangs and 5'phosphates and 3'hydroxyl groups can be precisely repaired by NHEJ [Clikeman et al, 2001; Lin et al, 1999].

The repair of DSBs through NHEJ requires three enzymatic activities; nucleases to remove damaged DNA, polymerases to aid in the repair and a ligase to restore the backbone (Fig. 2) [Lieber, 2008]. In mammalian cells the MRE11/RAD50/NBS1 (MRN) complex is also involved, preceding the binding of Ku [Shravastay et al, 2008] but it does not seem to be required for NHEJ [Rodrigue et al, 2006; Yang et al, 2006]. When Artemis (ATM) is lacking in cells, DNA-PK can phosphorylate histone H2AX, termed γ-H2AX [Burma et al, 2001; Collis et al, 2005; Cui et al, 2005; Shao et al, 1999; Burma and Chen, 2004; Chan et al, 1999; Karmaker et al, 2002; Yannone et al, 2001; Stucki and Jackson, 2006]. Since NHEJ simply ligates DNA ends without the use of a homologue chromosome to fix the DSBs, the local DNA does not return to its original sequence which explains the wide range of results [Shravastay et al, 2008].

Homologous recombination (HR)

The genomic stability of somatic cells is maintained through precise repair of double-stranded DNA breaks induced by exogenous and endogenous agents, such as damaged replication forks and repair of incomplete telomeres using a mechanism called homologous recombination (HR) [San Filippo et al, 2008]. HR only occurs during the S-phase and G2-phase of the cell cycle, before mitosis takes place [Alberts et al, 2008]. It is considered to be more precise than NHEJ because of the usage of homologous chromosomes, sister chromatids or repeated regions in the genome in order to restore the double-stranded breaks and can be up to one hundred percent accurate [Shrivastay et al, 2008; Brugmans et al, 2007] even though there are indications from studies observing HR for DSBs in yeast that point mutations adjacent to the site of repair are more frequent [Strathern et al, 1995]. Another problem for HR is that, with the exception of sister chromatids, the templates used for the repair are often not entirely homologous, which can result into loss of heterozygosity due to gene conversion [Nickoloff, 2002].

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The process of homologous recombination in order to repair DSBs involves several steps (Fig. 3), namely: end resection, strand invasion, synthesis, ligation, branch migration and holiday junction (HJ) resolution [Sharan and Kuznetsov, 2007; San Filippo et al, 2008]. A number of the involved proteins include RAD51, the MRE11/RAD50/NBS1 (MRN) complex, BRCA2, RPA, Dmc1, PALB2, DSS1, RAD52, and RAD54 [San Filippo et al, 2008; Shrivastay et al 2008; Czornak et al, 2008]


After the DSB has been formed, homologous recombination is initiated with end-processing at the broken ends [San Filippo et al, 2008], regulated by the MRN complex (the yeast homolog is called MRE11/RAD50/XRS2 - MRX - complex) and another exonuclease [Krogh and Symington, 2004; Shrivastay et al, 2008; San Filippo et al, 2008], resulting in 3' single-stranded DNA (ssDNA) tails. A recombinase filament is formed on the ssDNA ends, after which the homologous DNA is invaded to form a D-loop. DNA polymerases that have yet to be identified [Maolisel et al, 2008] extend the 3' end of the invading strand after allowing base-pairing of the invading and the complementary strands. The second DSB end is annealed to the extended D loop and the two crossed strands form Holliday junctions (HJs) [Holliday, 1964]. These 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 [Shrivastay et al, 2008; Czornak et al, 2008; Sharan and Kuzentsov, 2007; San Filippo et al, 2008; Liu and West, 2004; Holliday, 1964; Mizuuchi et al, 1982].

There also appears to be a role for BRCA1 as it interacts with BRCA2 and several other proteins that are considered repair factors, but as of yet the exact role is unclear in HR as well as NHEJ [Huang and D'Andrea, 2006].

In addition, homologous recombination plays an important role in meiosis by mediating an exchange of genetic information between the paternal and maternal alleles within the gamete precursor cells, thereby ensuring genetic diversity among the offspring of common parent and ensures proper segregation at the first meiotic division via cross over [Neale and Keeny, 2006; San Filippo et al, 2008].

MRE11/RAD50/NSB1 (MRN) complex

The MRN complex, consisting of the MRE11, RAD50 and NBS1 proteins is an important element in DNA replication, repair and signaling to the cell cycle checkpoints. In order to repair the damaged DNA, several kinases such as ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad-3-related (ATR) and DNA protein kinase catalytic subunit (DNA PKcs) phosphorylated protein targets. If the damage is irreparable these kinases direct the cell to programmed cell death, apoptosis [Shiloh, 2003a; Abraham, 2004]. Together with the MRN complex they are involved in genomic stability as well as telomere maintenance, and mutations in the genes encoding for these proteins lead to severe disorders that affect the repair mechanism [Czornak et al, 2008; Jazayeri et al, 2008].

The MRN complex is involved in both of the two major reparation mechanisms [D'Amours and Jackson, 2002; Van den Bosch et al, 2003], but primarily functions in cells with DNA double-strand breaks that have already undergone replication and thus will be repaired by homologous recombination [Stracker et al, 2004; D'Amours and Jackson, 2002]. ATM serine/threonine kinase is an important initiator of DNA damage response and is recruited and activated by MRN when there are DNA DSBs [Maser et al, 1997; Nelms et al, 1998; Difilippantonio et al, 2005; Uziel et al, 2003; Lee and Paull, 2004; Lee and Paull, 2005] but there are indications that ATM might be activated independent of the MRN complex [Difilippantonio et al, 2005], after which the MRN complex will bind to the broken ends of double-stranded DNA breaks due to MRE11 and RAD50 [Maser et al, 1997; De Jager et al, 2001b; Hopfner et al, 2002]. This association between RAD50 and MRE11 during the DNA processing stimulates the exonuclease and endonuclease activities of MRE11 [Moreno-Herrero et al, 2005; Paull and Gellert, 1999; Trujillo and Sung, 2001] and forming dimers it can bridge two DNA ends during HR-mediated double-strand breaks repair [Stracker et al, 2004; D'Amours and Jackson, 2002; Williams et al, 2008].

The DNA binding property of RAD50 is possibly involved in tethering sister chromatids during homologous recombination [Anderson et al, 2001; De Jager M et al, 2001; Wiltzius et al, 2005; Hopfner et al, 2002; Moreno-Herrero et al, 2005]. The proteins of the MRN complex are phosphorylated by ATM in response to double-stranded DNA breaks and this could modulate their functions in DSBs responses [Matsuoka et al, 2007; Gatei et al, 2000; Wu et al, 2000; Lim et al, 2000]. Moreover, Rad50, NBS1 and MRE11 each have their own properties and together they function as a holocomplex with an important role in the repair of double-stranded DNA breaks. However, it is still unclear what the precise role in non-homologous end joining is for the MRN complex, considering the NHEJ repair activity was not affected in Xenopus laevis by the addition of MRN [Huang and Dynan, 2002; Di Virgilio and Gautier, 2005] even though a deficient MRN complex after V(D)J recombination does lead to more unrepaired coding ends due to DSBs in developing G1-phase lymphocytes, which is very similar to ATM-deficient lymphocytes [Helmink et al, 2009].

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The entire complex is also capable of partly unwinding and dissociating the 3' overhang of the DNA duplex, which is probably one of the reasons the MRN complex is successful in processing double-stranded breaks repair.

It is difficult to discern the exact sequence of events because the activation of ATM and recruitment of the MRN complex take place rather fast, but considering the MRN complex has the quality to sense DNA damage it is upstream of ATM activation [Lee and Paull, 2004]. However, it has been shown that the MRN complex and ATM are interdependent when recognizing and signaling of double-stranded breaks [Lavin, 2007].

The fact that recruitment of ATM takes place in short time suggests that it might be partly activated before localization of DSBs [Bakkenist and Kastan, 2003], but the partially activated ATM cannot phosphorylated MRN-dependent substrates such as BRCA1 [Kitagawa et al, 2004]. It is possible that CtlP, a cell cycle regulation protein provides the MRN complex and BRCA1 with a physical connection [Limbo et al, 2007].

NSB1 only stimulates endonuclease activity [Paull and Gellert, 1999; Trujillo and Sung, 2001] and the gene that encodes for this protein, NBN, is possibly a tumor suppressor gene due to the effects that people with Nijmegen breakage syndrome (NSB) suffer but this has yet to be proven [Dzikiewicz-Krawczyk, 2008]. However, people with NSB do have problems with their DNA repair mechanisms because their MRN complex does not function properly, leaving them prone to DSBs [INBSSG, 2000; Digweed and Sperling, 2004].

Cancer and other deviations

A proteomic analysis has shown that there over 700 proteins involved in the DNA repair mechanism and all of them were phosphorylated by ATR kinases and ATM in response to DNA damage [Matsuoka et al, 2007]. Substrates of both ATM and ATR kinases influence and engage in cell cycle regulation, metabolism, structure and proliferation as well as signal transduction, immunity and oncogenesis and therefore it is suffice to say that dysfunction of either kinase can lead to serious disorders leaving one vulnerable to DNA damaging agents and DSBs. The MRN complex obviously plays a pivotal role in repairing double-stranded DNA breaks, whether that is through NHEJ or HR, and this is supported by the fact that dysfunction of particular proteins involved in these mechanisms lead to severe disorders. Ataxia telangiectasia (A-T), A-T-like disorder (ATLD), Nijmegen breakage syndrome (NBS) and NBS-variant are prime examples of such disorders.

A-T is an autosomal disorder, rare as well as recessive and is caused by mutations in the ATM gene and leads to, amongst others, radio sensitivity, chromosomal translocations, immunodeficiency and cancer predisposition because of total loss of ATM [Savitsky et al, 1995; Mavrou et al, 2008]. In vivo studies have shown that ATM is an inactive dimer that dissociates into active monomers once double-strand breaks are induced by ionizing radiation [Bakkenist and Kastan, 2003; Shiloh, 2006].

Mutations in MRE11 is responsible for ATLD and bi-allelic mutations are extremely rare [Stewart et al, 1999; Delia et al, 2004; Fernet et al, 2005]. Patients with this disorder are very sensitive to radiation and rearrangements involving chromosomes 7 and 14, but at a lower rate than in A-T [Stewart et al, 1999]. Considering the rarity of this disorder not a lot of research has been done and with minimal patients and that is also the reason why there is a lack of knowledge of the consequences of ATLD, because so far there are no immune deficiencies or malignancies reported for those patients [Taylor et al, 2004] but, like A-T, they do suffer from hypersensitivity to ionizing radiation and genomic instability [Hernandez et al, 1993; Klein et al, 1996; Stewart et al, 1999].

NBS is rare, caused by mutations in the NBN gene and is also an autosomal recessive chromosome instability disorder [Carney et al, 1998; Varon et al, 1998]. It is characterized by microcephaly, immunodeficiency, growth retardation and a very high cancer incidence, probably due to its involvement in cell cycle checkpoints and DNA damage response proteins [Becker et al, 2006]. A number of malignancies, like melanoma [Debniak et al, 2003; Steffen et al, 2006], non-Hodgkin lymphoma [Steffen et al, 2004; Chrzanowska et al, 2006; Steffen et al, 2006], acute lymphoblastic leukemia [Varon et al, 2000; Resnick et al, 2003; Chrzanowska et al, 2006], breast cancer [Gorski et al, 2005; Kanka et al, 2007; Sokolenko et al, 2007; Bogdanova et al, 2008], and prostate cancer [Cybulski et al, 2004; Hebbring et al, 2006], have been observed more frequently in heterozygote carriers of the NBN founder mutation. There is also overlap between NBS, A-T and ATLD [INBSSG, 2000; Digweed and Sperling, 2004] and it is estimated that about 200 people worldwide suffer from this disorder [Varon et al, 1998]. The mutation leads to two proteins, NBNp26 and NBNp70 and while the shorter fragment, NBNp26 does not seem to be associated with the MRE11 complex, the longer fragment is associated with it [Maser et al, 2001].

The NBS-variant is caused by mutations in the RAD50 gene [Gennery, 2006] and so far only one patient with two germline mutations has been reported [Bendix-Waltes et al, 2005]. The symptoms of the disorder are very similar to NBS but without the immunodeficiency [Gennery, 2006].

BRCA1 is a breast cancer tumor suppressor protein and has a role in NHEJ that possibly involves chromatin remodeling through the Fanconi anemia ubiquitylation pathway [Huang and D'Andrea, 2006] or MRE11 modulation [Durant and Nickoloff, 2005]. There is an alternative Ligase III-mediated NHEJ pathway involving PARP-1 but this is even more error-prone than the classical NHEJ [Wang et al, 2006].

Null mutations in one of the three components of the MRN complex lead to embryonic lethality in mice [Xiao and Weaver, 1997; Luo et al, 1999; Zhu et al, 2001] because of the resulting instable genome due to lacking of proper working DSBs repair mechanisms and mutations in the ATR genes leads to Seckel syndrome [O'Driscoll et al, 2003]. In addition, there are other syndromes associated with defects of other members in the DNA damage repair machinery, such as Artemis deficiency [Moshouse et al, 2001], Bloom syndrome (BS), Werner syndrome (WS) [Taniguchi and D'Andrea, 2006] and DNA ligase IV deficiency (LigIV) [O'Driscoll et al, 2001].

Patients with mutations in the BRCA2 gene are predisposed to breast, ovarian and other types of cancer [Jasin, 2002]. A cancer-associated truncation or BRCA2 impairs the transport of RAD51, an important protein involved in HR, to the nucleus [Davies et al, 2001]. Furthermore, it has been shown that BRCA2 binds DNA [Yang et al, 2005; San Filippo et al, 2006; Yang et al, 2002; Martin et al, 2005], is needed for DNA damage-induced nuclear RAD51 foci [Jasin, 2002; Yuan et al, 1999] and physically interacts with RAD 51 [Jasin, 2002; Sharan et al, 1997; Chen et al, 1998; Wong et al, 1997].


Double-strand DNA breaks (DSBs) are dangerous if not properly repaired. Thankfully, there are several repair mechanisms, the two major ones being non-homologous end joining (NHEJ) and homologous recombination (HR). Both machineries have their advantages but NHEJ seems to be more error-prone than HR under most circumstances. However, different circumstances call for different approaches as well as different mechanisms. The MRE11/RAD50/NSB1 (MRN) complex is involved in both NHEJ and HR and obviously plays a pivotal role in the repair of double-strand DNA breaks and without a proper working MRN complex all kinds of severe disorders arise and most include a predisposition for various types of cancer due to the lack of repair of DSBs after damaging the DNA with exogenous or endogenous agents.

In combination with ATM the MRN complex and their transducer and mediator proteins forms an efficient network that senses and signals DNA damage and activates the proper repair mechanisms. Considering this highly branched network depends on several proteins and kinases in order to repair DSBs, a defective key role component can have disastrous consequences, such as instable genome disorders as well as making one prone to various types of malignancies as there is a clear association between DNA damage and cancer.

The exact mechanism of DNA sensing is still unclear, even if there are several hypotheses. In addition, the precise role of the MRN complex in NHEJ still eludes us as well as how the choice between NHEJ and HR is regulated, considering NHEJ can occur at any time during the cell cycle. It is unknown what happens to the MRN complex after the DSBs are repaired.