**6. Molecular organization of the RAD51 recombinase**

RAD51 recombinase is an essential protein in HRR pathway that catalyzes strand transfer between a broken DNA and its undamaged homologous strand, allowing damaged region to be repaired [Thacker, 2005] Strand exchange reaction is initiated by RAD51-coating of ssDNA released from DSBs, to generate a nucleoprotein filament. This active thread binds the intact dsDNA substrate, searching and locating homologous sequences, and promoting DNA strand exchange in an ATP-dependent manner, forming a heteroduplex structure [Paques & Haber, 1999]. After DNA damage, RAD51 protein has been observed in nuclear complexes forming discrete foci, which are considered as the recombinational DNA repair

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]

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

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 &

RAD51 recombinase is an essential protein in HRR pathway that catalyzes strand transfer between a broken DNA and its undamaged homologous strand, allowing damaged region to be repaired [Thacker, 2005] Strand exchange reaction is initiated by RAD51-coating of ssDNA released from DSBs, to generate a nucleoprotein filament. This active thread binds the intact dsDNA substrate, searching and locating homologous sequences, and promoting DNA strand exchange in an ATP-dependent manner, forming a heteroduplex structure [Paques & Haber, 1999]. After DNA damage, RAD51 protein has been observed in nuclear complexes forming discrete foci, which are considered as the recombinational DNA repair

**6. Molecular organization of the RAD51 recombinase** 

(**Fig. 5A**).

[Hopfner et al., 2002].

Gellert, 1999].

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

DNA Repair in Pathogenic Eukaryotic Cells:

SIP-IPN (Mexico) and European Community grants.

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(October 2002), pp. 1385–1392. ISSN 0020-7519.

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147. ISSN 0166-6851

**8. Acknowledgments** 

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Insights from Comparative Genomics of Parasitic Protozoan 383

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rectangles) and DNA-binding domains (gray rectangles). **(B)**. Functional and structural domains of RAD50 proteins. Walker A (N terminus) and Walker B (C terminus) are marked in the ABC-ATPase domains (gray rectangles), while MRE11-binding sites flank Walker motifs (dark gray rectangles) and the CysXXCys hook domain (white rectangles) are labeled in the coiled-coil central regions. (**C)**. Functional and structural domains of RAD51 proteins. Polymerization motif (PM), as well as Walker A and B motifs, L1 and L2 regions and ATP cap appear as colored rectangles. Boxshade panels: black boxes, identical aa; grey boxes, conserved substitutions.

sites [Tashiro et al., 2000]. RAD51 proteins have been identified in *Trypanosoma brucei* and *Plasmodium falciparum* parasites, which perform HRR to switch the expression of genes encoding surface membrane glycoproteins and generate antigenic variation [Conway et al., 2002; Freitas-Junior et al., 2000]. Furthermore, recombinational rearrangements are responsible for amplification of the multidrug resistance *pfmdr1* gene in *P. falciparum* [Triglia et al., 1991] demonstrating the relevance of HRR to generate genomic versatility and plasticity in protozoan parasites. Molecular analysis and functional assays confirmed that recombinant EhRAD51 is a *bonafide* recombinase that is able to catalyze specific ssDNA transfer to homologous dsDNA forming the three-stranded pairing molecule called Dloop structure. In addition, *E. histolytica RAD51* sequence conserves the typical architecture of RECA/RAD51 family members [Lopez-Casamichana et al., 2008]. Amino acid sequences multiple alignment of RAD51 orthologues from *E. histolytica, S. cerevisae, T. vaginalis*, *G. lamblia* and *P. falciparum* revealed that all these proteins share functional and structural conserved motifs (**Fig. 5C**). Each of them contains the putative polymerization motif (PM), which tethers individual subunits to form quaternary assemblies in human RAD51 protein [Bell, 2005]. We also identified the ATPase Walker A or phosphate binding loop (P-loop) and Walker B motifs residues, the ssDNA binding loops L1 and L2, as well as the ATP stacking motif or ATP cap at the C terminus, which are essential for nucleofilament assembling and ATP hydrolysis in RAD51/RECA-like recombinases [Shin et al., 2003; Conway et al., 2004].
