**5. Molecular organization of the MNR complex**

The MRE11–RAD50–NBS1 (MRN) complex is considered to have an imperative function in DSB repair. This protein complex operates as DSB sensor, co-activator of DSB-induced cell cycle checkpoint signaling, and as a DSB repairs effector in both the HRR and NHEJ pathways [Taylor et al., 2010; Rass et al., 2009]. Additionally, it has also been found to associate with telomeres maintenance at the ends of linear chromosomes. MRE11 and RAD50 orthologues have been reported in all taxonomic Kingdoms. MRE11, RAD50, and XRS2 (the *S. cerevisiae* homologue of vertebrate-specific NBS1) were initially recognized through yeast resistance to DNA damage induced by UV light and X-rays and meiotic recombination studies [Ogawa et al., 1995]. To efficiently perform these functions, this complex has shown particular enzymatic roles. Biochemical experiments have revealed that the phosphoesterase domain of MRE11 works as both a single-and double-stranded DNA endonuclease, besides as 3´–5´ dsDNA exonuclease [D'Amours & Jackson, 2002]. Furthermore, RAD50 and NBS1/Xrs2 are able to promote the activity of MRE11, in an ATP dependent manner [Paul & Gellert, 1998]. ATP binding by RAD50 stimulates the binding of the MR complex to 3´ overhangs and, also, ATP hydrolysis is required to arouse the cleavage of DNA hairpins, inducing modification of endonuclease specificity via DNA relaxing [Paull & Gellert, 1998; de Jager et al., 2002].

Fig. 3. **Phylogenetic relationships between RAD3 from** *S. cerevisiae***,** *E. histolytica***,** *T. vaginalis***,** *G. lamblia* **and** *P. falciparum***.** The unrooted tree was created with the MEGA 5.05 program using the Neighbor Joining algorithm based on ClustalW. Numbers above the tree nodes indicate the percentage of times that the branch was recovered in 500 replications.

The MRE11–RAD50–NBS1 (MRN) complex is considered to have an imperative function in DSB repair. This protein complex operates as DSB sensor, co-activator of DSB-induced cell cycle checkpoint signaling, and as a DSB repairs effector in both the HRR and NHEJ pathways [Taylor et al., 2010; Rass et al., 2009]. Additionally, it has also been found to associate with telomeres maintenance at the ends of linear chromosomes. MRE11 and RAD50 orthologues have been reported in all taxonomic Kingdoms. MRE11, RAD50, and XRS2 (the *S. cerevisiae* homologue of vertebrate-specific NBS1) were initially recognized through yeast resistance to DNA damage induced by UV light and X-rays and meiotic recombination studies [Ogawa et al., 1995]. To efficiently perform these functions, this complex has shown particular enzymatic roles. Biochemical experiments have revealed that the phosphoesterase domain of MRE11 works as both a single-and double-stranded DNA endonuclease, besides as 3´–5´ dsDNA exonuclease [D'Amours & Jackson, 2002]. Furthermore, RAD50 and NBS1/Xrs2 are able to promote the activity of MRE11, in an ATP dependent manner [Paul & Gellert, 1998]. ATP binding by RAD50 stimulates the binding of the MR complex to 3´ overhangs and, also, ATP hydrolysis is required to arouse the cleavage of DNA hairpins, inducing modification of endonuclease specificity via DNA

**5. Molecular organization of the MNR complex** 

relaxing [Paull & Gellert, 1998; de Jager et al., 2002].

In this chapter, we have identified the presence of *Mre11* and *Rad50* genes in the genome of *E. histolytica*, *T. vaginalis*, *G. lamblia* and *P. falciparum.* However, all analyzed pathogenic eukaryotic cells, with the exception *of E. histolytica,* lack the *Xrs2* homologue. The absence of a NBS1/Xrs2 homologous sequence in the other parasites might seem antagonistic to the idea of the existence of an active MRN complex. However we cannot discard the possibility that these microorganisms use a very divergent NBS1 protein, or even that this third component could be unessential. In order to initiate the characterization of components of MRN complex in these parasites, we studied the structural and evolutionary relationships between MRE11, RAD50 and NBS1 through PSI-BLAST analysis in comparison to human and yeast orthologues. This program generates a weighted profile from the sequences detected in the first pass of a gapped-BLAST search and iteratively searches the database using this profile as the query, allowing the inclusion of sequences with e-value cut off higher than 0.01 [Alschult et al., 1997]. Using the e-value threshold as a similarity measure, we evidenced a close relation between putative EhMRE11, HsMRE11, ScMRE11, TvMRE11 and PfMRE11. Conversely, GlMRE11 turned out to be less similar to the others, being closer to *E. histolytica* and *T. vaginalis* proteins (**Fig. 4**). On the other hand, analysis of RAD50 orthologues exposed a great conservation of these proteins, since all e-value threshold were <0.0001. As we have previously reported, EhNBS1 is closer to its human homologue than yeast [Lopez-Casamichana et al., 2007].

Fig. 4. **Individual protein relationships of MRN complex in pathogenic eukaryotic cells.** Similarity was evaluated through PSI-BLAST analysis. The width of connecting lines indicates similarity level.

DNA Repair in Pathogenic Eukaryotic Cells:

Insights from Comparative Genomics of Parasitic Protozoan 381

Fig. 5. **Comparison of the amino acids sequence of MRE11, RAD50 and RAD51 proteins of** *S. cerevisae E. histolytica, T. vaginalis***,** *P. falciparum* **and** *G. lamblia***. (A)**. Functional and structural domains of MRE11 proteins. MRE11 phosphoesterase motifs I-V (black

To better understand the functionality of MRN complex in these parasites, predicted amino acid sequences of RAD50 and MRE11 were compared through multiple alignment using ClustalW software (http://www.ebi.ac.uk/ clustalw/). Reported functional and structural domains were surveyed using Prosite (http://www.expasy.org/tools/scanprosite/), Pfam (http://www.sanger.ac.uk /Software/Pfam/), SMART (http://smart.emblheidelberg.de/) and Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motifscan) programs. For all studied parasites, our search revealed that the MRE11 orthologues contain the N-terminal Mn2+/Mg2+-dependent nuclease domain including the five conserved phosphoesterase motifs described in yeast protein [Hopkins & Paull, 2008. Moreover, C-terminal DNA binding domains were also identified [Williams et al., 2007; D'Amours & Jackson, 2002] (**Fig. 5A**).

RAD50 proteins displayed sequence and organizational homology to structural maintenance of chromosome (SMC) family members that control the higher-order structure and dynamics of chromatin. The N-terminal Walker A and C-terminal Walker B nucleotide binding motifs, which associate one with another to form a bipartite ATP-binding cassette (ABC)-type ATPase domain, were predicted [Hopfner et al., 2000; Hopfner et al, 2001]. Furthermore, amino acids flanking Walker motifs form coiled-coil configurations that converge with the cysteine zinc hook (CysXXCys) motif [Hopfner et al., 2002] (**Fig. 5B**). In the interphase of Walker domains, there are two MRE11 binding sites. Formation of the stable MRE11-RAD50 complex is reached by each unit of the MRE11 dimer binding a RAD50 molecule at the intersection of its globular and coiled-coil domains [de Jager et al., 2001a]. Scanning force microscopy experiments have demonstrated that whereas the globular head of the Mre112Rad502 complex links with the ends of linear dsDNA, the two coiled-coil regions of RAD50 are stretchy ''arms", and project outward away from the DNA [Hopfner et al., 2002].

The third member of the MRN complex is NBS1 protein that was only detected in *E. histolytica*, but not in *G. lamblia*, *P. falciarum* neither *T. vaginalis*. We have previously reported that EhNBS1 consists of an FHA domain and adjacent BRCT domains at its Nterminus [Lopez-Casamichana et al., 2007]. In *Homo sapiens*, the FHA domain binds phosphorylated threonine residues in Ser-X-Thr motifs present in DNA damage proteins, including CTP1 and MDC1. The BRCT domains in human NBS1 fix Ser-X-Thr motifs when the serine residue is phosphorylated. These phospho-dependent interactions are significant for recruiting repair machineries and checkpoint proteins to DNA DSBs [Lloyd et al., 2009; Williams et al., 2009]. In reconstitution studies, the affinity of MRE11-RAD50 for DNA and its nuclease activity is further enhanced by the addition of NBS1 [Paull & Gellert, 1999].
