**2. DNA repair machineries in pathogenic eukaryotic cells**

#### **2.1 Identification of DNA repair machineries**

In order to identify amino acids sequences of *E. histolytica*, *G. lamblia*, *P. falciparum* and *T. vaginalis* proteins related to DNA repair factors, we performed similarity searches in the Eupath database (http://eupathdb.org/eupathdb/) using the *Saccharomyces cerevisiae* DNA repair proteins from HRR, NHEJ, BER, NER and MMR machineries as probes [reviewed in Lopez-Camarillo et al., 2009]. Putative gene products were selected from BLAST analysis against each parasite database using the Blosum 62 scoring matrix and the following criteria: (i) at least 20% identity and 35% homology to the query sequence and (ii) e-value lower than 0.002, unless a portion of the protein showed a very strong similarity. All sequences, as well as the *E. histolytica* sequences obtained from previous work [López-Camarillo et al., 2009], were then verified by BLAST against *S. cerevisiae* and *Homo sapiens* databases to confirm their identity. Additionally, we also retrieved data from published reports about *G. lamblia*,

DNA repair mechanisms, and genome evolution. *Entamoeba histolytica* and *Giardia lamblia* (syn. *G. intestinalis, G. duodenalis*) are intestinal parasites that cause diarrheal diseases. *E. histolytica* is responsible for fulminating dysentery, bloody diarrhea, weight loss, fatigue, abdominal pain, which affect 50 million people and provoke 100,000 deaths in developing countries each year. In some cases, *E. histolytica* trophozoites can cross the intestinal wall and use the blood stream to reach different vital organs of the human body, usually the liver (but also the lungs, brain or spleen) to provoke liver abscesses, which can be fatal if untreated [Guo et al., 2007]. *G. lamblia*  is another contributor to the enormous burden of diarrheal diseases with over 250 million symptomatic human infections per year worldwide. This anaerobic flagellated protozoa colonises and reproduces in the small intestine of several vertebrates, including human, causing giardiasis, commonly known as Beaver fever, which is characterized by diarrhea, excess gas, stomach or abdominal cramps, upset stomach, and nausea. Additionally, *Giardia* infection has an adverse impact on child linear growth and psychomotor development since the parasite causes iron-deficiency anemia, micronutrient deficiencies and growth retardation associated with diarrhea and malabsorption syndrome [Ankarklev et al., 2010]. Individuals become infected by *E. histolytica* and *G. lamblia* through ingesting or coming into contact with food, soil, or water that have been contaminated by the feces of an infected human or animal. *Plasmodium falciparum* is the protozoan parasite responsible for human malaria, which is one of the most severe infectious diseases with 240 million cases in 2009 and more than 1 million deaths in children each year in Africa alone. The presence of the parasite in red blood cells lead them to stick to blood vessels through a process called cytoadherence, which produce the obstruction of the microcirculation and dysfunction of multiple organs, typically the brain in cerebral malaria. Symptoms usually include fever and headache, in severe cases progressing to coma, and death (Kokwaro, 2009). Trichomoniasis caused by *Trichomonas vaginalis* is the most common nonviral sexually transmitted disease (STD) in the world [WHO, 1995]. It has long been recognized as a frequent cause of vaginitis in women and urethritis in men, but data now link it to cervical cancer and bad pregnancy outcomes [Cotch et al., 1997], as well as to an enhanced risk for human immunodeficiency virus transmission [Sorvillo & Kerndt , 1998]. Here, we combined the use of genomic approaches based on bioinformatic analysis of parasite genome sequence with the review of published reports to perform a comparative description of DNA repair machineries from *E. histolytica*, *G. lamblia*, *P. falciparum* and *T. vaginalis*, which

cause high morbidity and mortality in many developed and developing countries.

In order to identify amino acids sequences of *E. histolytica*, *G. lamblia*, *P. falciparum* and *T. vaginalis* proteins related to DNA repair factors, we performed similarity searches in the Eupath database (http://eupathdb.org/eupathdb/) using the *Saccharomyces cerevisiae* DNA repair proteins from HRR, NHEJ, BER, NER and MMR machineries as probes [reviewed in Lopez-Camarillo et al., 2009]. Putative gene products were selected from BLAST analysis against each parasite database using the Blosum 62 scoring matrix and the following criteria: (i) at least 20% identity and 35% homology to the query sequence and (ii) e-value lower than 0.002, unless a portion of the protein showed a very strong similarity. All sequences, as well as the *E. histolytica* sequences obtained from previous work [López-Camarillo et al., 2009], were then verified by BLAST against *S. cerevisiae* and *Homo sapiens* databases to confirm their identity. Additionally, we also retrieved data from published reports about *G. lamblia*,

**2. DNA repair machineries in pathogenic eukaryotic cells** 

**2.1 Identification of DNA repair machineries** 

*P. falciparum* and *T. vaginalis* (**Table 1**). The absence of a given sequence in the table indicates that the corresponding gene was not identified in the parasite genome or that the sequence was too divergent to be detected by our *in silico* strategy.

None of the protozoan parasites studied here has the complete DNA repair pathways reported in yeast. HRR is the most conserved pathway suggesting that it is the mayor DSB repair pathway in these protozoan parasites. *E. histolytica*, *G*. *lamblia*, *P. falciparum* and *T. vaginalis* genomes contain most of the RAD52 epistasis group genes, although their functional relevance remains to be determined. Homologs for RAD50, RAD51, MRE11, RAD54 and RPA (lacking the RAD52 interacting domain) have been previously reported in *P. falciparum* [Voss et al., 2002; Malik et al., 2008]. In agreement with its participation in DNA repair, the *PfRad51* gene is overexpressed in the mitotically active schizont stage and in response to methyl methane sulfonate [Bhattacharyya & Kumar, 2003]. In. *T. vaginalis*, RAD50 y MRE11 were previously published as components of the meiotic recombination machinery, although meiosis has not been observed in this organism [Malik et al., 2008]. Ramesh et al. [2005] and Malik et al. [2008] identified the *Rad50/Mre11*, *Rad52* and *Dmc1 genes* involved in meiotic recombination machinery by HRR in *Giardia*. Intriguingly, *G. lamblia* and *P. falciparum* lack the *nsb1* homologue (*xrs2* in Yeast) that is a component of the MRN complex involved in DSB detection and 3´ ssDNA tails conversion. Recently, we published the *E. histolytica* RAD52 epistasis group involved in HRR [Lopez-Casamichana et al., 2007, 2008]. Interestingly, RT-PCR assays evidenced that some genes were down-regulated, whereas others were up-regulated when DSB were induced by UV-C irradiation, which revealed an intricate transcriptional modulation of *E. histolytica* RAD52 epistasis group related genes in response to DNA damage. Particularly, *Ehrad51* mRNA expression was 16-, 11- and 4-fold increased at 30 min, 3 h and 12 h, respectively. DNA microarrays assays confirmed the activation of *EhMre11, EhRad50*, and *EhRad54* genes at 5 min after DSB induction, suggesting that they represent early sensors of damage in HRR pathway [Weber et al., 2009]. Additionally, the molecular characterization of EhRAD51 showed that the presence of all the functional domains reported in yeast and human homologues. EhRAD51 was upregulated and redistributed from cytoplasm to the nucleus of trophozoites at 3 h after DNA damage and it was able to catalyze specific single-strand DNA (ssDNA) transfer to homologous double strand DNA (dsDNA) forming the three-stranded pairing molecule called D-loop structure, confirming that it is a *bonafide* recombinase in *E. histolytica* [Lopez-Casamichana et al., 2008].

*G. lamblia* and P*. falciparum* only have three of the eight factors of the NHEJ pathway (including the MNR complex also involved in HRR), which strongly suggest that they preferably use HRR to repair DSB. In contrast, almost all NEHJ pathway factors have been identified in *E. histolytica* and *T. vaginalis*, including the LIF1 ligase, RAD27 nuclease and MRE11/RAD50/NSB1 proteins. However, *E. histolytica* genome does not contain a homologous gene for KU80 subunit [López-Camarillo et al., 2009] and *T. vaginalis* lacks both *ku70* and *ku80* genes [Carlton et al., 2007]. As these proteins form a single KU complex that recognizes DSB sites and recruits other DNA repair factors, our findings could appear contradictory. The absence of conserved KU proteins has also been reported in *Encephalitozoon cunili* [Gill & Fast, 2007] and yeast [Hefferin & Tomkinson, 2005], thus it is possible that these organisms use highly divergent KU proteins to perform the NHEJ pathway.

The other key DNA repair mechanisms represented by BER, NER and MMR pathways operate to repair aberrant bases or nucleotides from a ssDNA using the complementary strand as template for DNA synthesis. As in *E. histolytica* [Lopez-Camarillo et al., 2009], the *G. lamblia* BER pathway appears to be largely incomplete, lacking *apn1*, *mag1, ogg1*, *rad10*, *mus81* and *mms4* genes. Both parasites live under oxygen-limiting conditions and have a

DNA Repair in Pathogenic Eukaryotic Cells:

*sgs1* C4M4V5 A8BAJ1


**Non homologous end joining (NHEJ) pathway** 

*rad27* C4M6G8 A8B672

C4LYM7

**Nucleotide excision repair (NER) pathway** 

C4M8Q4 C4M6T8

*rad3* C4M8K7

*pcna* C4M9R9 A8BIU1 P61074

**Base excision repair (BER) pathway** 

*ntg1* C4M764

C4MBG9 -

rad17 ddc1 mec3

*ku80 ku70* 

*rpa1* C4M8G6 - Q9U0J0

Insights from Comparative Genomics of Parasitic Protozoan 373

Q8I3A1

Q8I2W7 Q8ILG5

> - - -

> - -

Q7K734 Q8IJW1

Q7KQJ9

A2G5D0 P22336

A2DYY2 P35187

A2GNP0 P26793

A2DQV2 P15873

P06839

P48581 Q08949 Q02574

P32807 Q04437


> - -


Q8I2H7 A2G2G8

A2E4I6 A2F1W2 A2DDD4 A2E1B9 A2ELX1 A2G2G9\*

**Gene name** *E. histolytica G. lamblia P. falciparum T. vaginalis S. cerevisiae rad59* - - - - Q12223 *exo1* C4MBM5 A8BQ11 Q8IBK1 A2E2N7 P39975

*rpa2* C4LT79 - - - P26754

*rad24* C4M5T7 - - A2D9F4 P32641 *hpr5* - - Q8I3W6 A2F783 P12954

*lif1* - - - - P53150 *dnl4* C4M5H3 - - A2DFX6 Q08387

*apn1* - - Q9BMG7 - P22936 *apn21* - A8BGE2 O97240 - P38207 *mag1* - - - - P22134 *ogg1* - - Q8I2Y2 - P53397

*ung1* C4LUV5 A8B632 Q8ILU6 A2GFQ7 P12887

*rad1* C4LT01 D3KH96 Q8ID22 A2DS24 P06777 *rad10* C4LW01 - O96136 A2DBF5 P06838 *cdc9* C4M5H3 A8BWV4 Q8IES4 A2DFX6 P04819 *mus81* - - - A2FKU9 Q04149 *mms4* - - - A2DHF7 P38257

*rad2* C4M0V9 - O96154 A2GNP0 P07276

A8BYS3 A8B495

A8B9Y0



D3KG58

highly reduced form of mitocondria called mitosomes [Tovar et al., 1999, 2003]. Then the absence of OGG1 could indicate that they do not suffer oxidative damage to mitochondrial DNA. In contrast, *Plasmodium* Flap endonuclease-1 (PfFEN-1) and Pf DNA Ligase I (PfLigI) have enzymatic activities similar to other species [Gardner et al., 2002; Casta et al., 2008], indicating that BER pathway should be functional in this parasite although several components are lacking.

Most genes involved in NER pathway are represented in *E. histolytica* [Lopez-Camarillo et al., 2009]*, G. lamblia, P. falciparum and T. vaginalis* genomes suggesting that this mechanism could be potentially active in these eukaryotic parasites. PfXPB/RAD25, PfXPG/RAD2 and PfXPD/RAD3 have been previously reported in *P. falciparum* [Gardner et al., 2002; Bethke et al., 2007; Casta et al., 2008]. Additionally, the overexpression of *EhDdb1, EhRad23* and *EhRad54* genes after UV-induced DNA damage in *E. histolytica* [Weber et al., 2009] suggested that these genes could be involved in chromatin remodeling complexes as their homologues in human and yeast. *E. histolytica*, *G. lamblia* and *T. vaginalis* have various *rad3* genes to form the NEF3 complex (RAD2, RAD3, RAD25) of the BER pathway. Particularly, we identified six *rad3* genes and an additional truncated gene in *T. vaginalis*. On the other hand, all the parasites studied here lack almost one of the components of the TFIIH complex subunits (TFB1, TFB2 or TFB3).

As in bacteria, *Drosophila melanogaster*, *H. sapiens* and many other organisms [Lisby & Rothstein, 2005], *E. histolytica*, *G. lamblia* [Ramesh et al., 2005], *P. falciparum* [Bethke et al., 2007] and *T. vaginalis* [Malik et al., 2008] have almost all *S. cerevisiae* MMR genes, including the components of the MUTS (MSH2/MSH6) heterodimer, which strongly suggest that MMR could be an active DNA repair pathway in these parasites. Notably, *E. histolytica* and *P. falciparum* have two *msh2* genes. However, neither *E. histolytica* nor *P. falciparum* present the *msh3* gene that is required for the formation of the MUTS (MSH2/MSH3) heterodimer. PfMSH2-1, PfMSH2-2, PfMSH6, PfMLH1 and PfPMS1 proteins potentially participating in MMR have been previously reported in *P. falciparum*. Inhibition of *PfMSH2-2* gene increased mutation rate and microsatellite polymorphism, indirectly demonstrating its relevance in MMR and microsatellite slippage prevention. Moreover, antimalarial drug resistance has been recently related to a defective DNA mismatch repair, mainly in PfMutLα content [Castellini et al., 2011], which demonstrated the relevance of this mechanism for the parasite biology.


highly reduced form of mitocondria called mitosomes [Tovar et al., 1999, 2003]. Then the absence of OGG1 could indicate that they do not suffer oxidative damage to mitochondrial DNA. In contrast, *Plasmodium* Flap endonuclease-1 (PfFEN-1) and Pf DNA Ligase I (PfLigI) have enzymatic activities similar to other species [Gardner et al., 2002; Casta et al., 2008], indicating that BER pathway should be functional in this parasite although several

Most genes involved in NER pathway are represented in *E. histolytica* [Lopez-Camarillo et al., 2009]*, G. lamblia, P. falciparum and T. vaginalis* genomes suggesting that this mechanism could be potentially active in these eukaryotic parasites. PfXPB/RAD25, PfXPG/RAD2 and PfXPD/RAD3 have been previously reported in *P. falciparum* [Gardner et al., 2002; Bethke et al., 2007; Casta et al., 2008]. Additionally, the overexpression of *EhDdb1, EhRad23* and *EhRad54* genes after UV-induced DNA damage in *E. histolytica* [Weber et al., 2009] suggested that these genes could be involved in chromatin remodeling complexes as their homologues in human and yeast. *E. histolytica*, *G. lamblia* and *T. vaginalis* have various *rad3* genes to form the NEF3 complex (RAD2, RAD3, RAD25) of the BER pathway. Particularly, we identified six *rad3* genes and an additional truncated gene in *T. vaginalis*. On the other hand, all the parasites studied here lack almost one of the components of the TFIIH complex subunits

As in bacteria, *Drosophila melanogaster*, *H. sapiens* and many other organisms [Lisby & Rothstein, 2005], *E. histolytica*, *G. lamblia* [Ramesh et al., 2005], *P. falciparum* [Bethke et al., 2007] and *T. vaginalis* [Malik et al., 2008] have almost all *S. cerevisiae* MMR genes, including the components of the MUTS (MSH2/MSH6) heterodimer, which strongly suggest that MMR could be an active DNA repair pathway in these parasites. Notably, *E. histolytica* and *P. falciparum* have two *msh2* genes. However, neither *E. histolytica* nor *P. falciparum* present the *msh3* gene that is required for the formation of the MUTS (MSH2/MSH3) heterodimer. PfMSH2-1, PfMSH2-2, PfMSH6, PfMLH1 and PfPMS1 proteins potentially participating in MMR have been previously reported in *P. falciparum*. Inhibition of *PfMSH2-2* gene increased mutation rate and microsatellite polymorphism, indirectly demonstrating its relevance in MMR and microsatellite slippage prevention. Moreover, antimalarial drug resistance has been recently related to a defective DNA mismatch repair, mainly in PfMutLα content [Castellini et al., 2011], which demonstrated the relevance of this mechanism for the parasite biology.

**Gene name** *E. histolytica G. lamblia P. falciparum T. vaginalis S. cerevisiae*

*rad50* C4M2L7 Q6WD96 C6KSQ6 A2FAD3 P12753

*nbs1* C4M874 - - A2DHF7 P33301

*rad52* C4M197 Q6WD95 - - P06778

Pf11\_0087\*\*

C4M5L7 - - A2GIB8 P38953

PFA0390w\*\* A2ECB0 P32829


A2FXT7 P25454

P25301

Q86CI9 A8BR27\*

**Homologous recombination repair (HRR) pathway** 

C4LVX7 C4M8N7\*

C4LVM6 C4M7S7

*rad51* C4M4K4 Q86C21 Q8IIS8

*mre11* Q86C23

*rad54 rad54b* 

*rad51c rad57* 

components are lacking.

(TFB1, TFB2 or TFB3).


DNA Repair in Pathogenic Eukaryotic Cells:

each graph.

**proteins** 

Insights from Comparative Genomics of Parasitic Protozoan 375

Fig. 1. **Conservation of DNA repair pathways between** *E. histolytica* **and** *G. lamblia* **(A),** *P. falciparium* **(B) and** *T. vaginalis* **(C).** Amino acids sequences from orthologous proteins were compared by Blast and the percentage of identity was determined through pair wise

alignment of the most conserved region. Average identity of all pathways is indicated above

**2.3 DNA repair activity in cell free lysates evidences the functionality of DNA repair** 

Although insights about the activity of DNA repair proteins in protozoa have been mainly obtained from experimental evidence based in heterologous expression and characterization of recombinant proteins, some reports showed that DNA repair activity could be detected in whole cell extracts, supporting the notion that DNA repair pathways already operates *in vivo*. For instance, Haltiwanger et al., (2000) reported the characterization of an AP


Table 1. Comparison of DNA repair machineries from *E. histolytica*, *G. lamblia*, *P. falciparum*, *T. vaginalis* and *S. cerevisiae*. \* fragment, \*\* PlasmoDB database.

#### **2.2 Conservation of DNA repair pathways**

To investigate the degree of conservation of DNA repair pathways in protozoan parasites, we next determined the values of Smith-Waterman identity scores between *E. histolytica* proteins and their corresponding orthologues in *G. lamblia*, *P. falciparum* and *T. vaginalis* by BLAST analysis based in pairwaise sequence alignments and calculated the mean value for each DNA repair machinery (**Fig. 1**). Data of the MNR complex which participate in HRR and NHEJ pathways were included in both mechanisms. DSB repair pathways were generally more conserved than Excision Repair mechanisms. Considering amino acids identity, mean values for HRR and NHEJ pathways were higher in *E. histolytica*/*P. falciparum* comparison, suggesting that *E. histolytica* machinery was closer to *P. falciparum* than to *G. lamblia* and *T. vaginalis* machineries. The comparison *E. histolytica*/*G. lamblia* evidenced that HRR is highly conserved between both parasites, whereas components of the other pathways were more divergent. In the case of *E. histolytica*/*P. falciparum* comparison, NHEJ appeared to be more conserved that HRR, while the identity of HRR and NHEJ factors was very similar in *E. histolytica*/*T. vaginalis*. In all the parasites, the RAD51 recombinase is the most conserved protein (51%, 58% and 64% when *E. histolytica* protein sequence was compared with *G. lamblia*, *P. faciparum* and *T. vaginalis* orthologues, respectively), which is consistent with its relevant role in HRR mechanism.

**Gene name** *E. histolytica G. lamblia P. falciparum T. vaginalis S. cerevisiae rad4* - - - - P14736 *rad7* - - - - P06779 *rad14* - - - - P28519 *rad16* - A8BL62 Q8I4S6 A2D9P9 P31244 *rad23* C4MAR5 D3KF29\* Q8IJS8 A2FM19 P32628 *rad25* C4MA19 A8BMI7 Q8IJ31 A2DEA8 Q00578 *rad26* C4MAR8 A8BK31 - A2EXQ4 P40352 *rad28* - - Q8IJ73 A2DZ24 Q12021 *ssl1* C4LV67 A8BA50 Q8IEG6 A2ENQ3 Q04673 *tfb1* C4LWV8 - - - P32776 *tfb2* C4MIG0 - Q8I4Y8 A2E2N2 Q02939 *tfb3* - D3KH94 Q8I3Y3 - Q03290 *tfb4* C4M9E2 A8B6C2 Q8IDG5 A2EYI3 Q12004

*mlh1* C4M5R1 - Q8IIJ0 A2EGR5 P38920

*msh3* - - - - P25336 *msh6* C4M4T8 A8BC61 Q8I447 A2EA54 Q03834 *pms1* C4LW71 A8B4I6 Q8IBJ3 A2G2B4 P14242 Table 1. Comparison of DNA repair machineries from *E. histolytica*, *G. lamblia*, *P. falciparum*,

Q6WD97 Q8ILI9

To investigate the degree of conservation of DNA repair pathways in protozoan parasites, we next determined the values of Smith-Waterman identity scores between *E. histolytica* proteins and their corresponding orthologues in *G. lamblia*, *P. falciparum* and *T. vaginalis* by BLAST analysis based in pairwaise sequence alignments and calculated the mean value for each DNA repair machinery (**Fig. 1**). Data of the MNR complex which participate in HRR and NHEJ pathways were included in both mechanisms. DSB repair pathways were generally more conserved than Excision Repair mechanisms. Considering amino acids identity, mean values for HRR and NHEJ pathways were higher in *E. histolytica*/*P. falciparum* comparison, suggesting that *E. histolytica* machinery was closer to *P. falciparum* than to *G. lamblia* and *T. vaginalis* machineries. The comparison *E. histolytica*/*G. lamblia* evidenced that HRR is highly conserved between both parasites, whereas components of the other pathways were more divergent. In the case of *E. histolytica*/*P. falciparum* comparison, NHEJ appeared to be more conserved that HRR, while the identity of HRR and NHEJ factors was very similar in *E. histolytica*/*T. vaginalis*. In all the parasites, the RAD51 recombinase is the most conserved protein (51%, 58% and 64% when *E. histolytica* protein sequence was compared with *G. lamblia*, *P. faciparum* and *T. vaginalis* orthologues,

C0H4L8

A2EP54 P25847

**Mismatch repair (MMR) pathway** 

B1N4L6

**2.2 Conservation of DNA repair pathways** 

*T. vaginalis* and *S. cerevisiae*. \* fragment, \*\* PlasmoDB database.

respectively), which is consistent with its relevant role in HRR mechanism.

*msh2* C4M9J9

Fig. 1. **Conservation of DNA repair pathways between** *E. histolytica* **and** *G. lamblia* **(A),** *P. falciparium* **(B) and** *T. vaginalis* **(C).** Amino acids sequences from orthologous proteins were compared by Blast and the percentage of identity was determined through pair wise alignment of the most conserved region. Average identity of all pathways is indicated above each graph.

#### **2.3 DNA repair activity in cell free lysates evidences the functionality of DNA repair proteins**

Although insights about the activity of DNA repair proteins in protozoa have been mainly obtained from experimental evidence based in heterologous expression and characterization of recombinant proteins, some reports showed that DNA repair activity could be detected in whole cell extracts, supporting the notion that DNA repair pathways already operates *in vivo*. For instance, Haltiwanger et al., (2000) reported the characterization of an AP

DNA Repair in Pathogenic Eukaryotic Cells:

1993; Gilbert et al., 1997].

**4. Duplicated genes: The case of** *rad3*

Insights from Comparative Genomics of Parasitic Protozoan 377

Gene duplicates represent for 8-20% of the genes in eukaryotic cells, and the rates of gene duplication are estimated at between 0.2% and 2% per gene per million years. Gene duplications are one of the major motors in the evolution of genetic systems and may occur in homologous recombination, retrotransposition event, or duplication of an entire chromosome [Zhang, 2003]. Duplicated genes are believed to be a main system for the establishment of new gene functions generating evolutionary novelty [Long & Langley,

A detailed examination of **Table 1** revealed that several DNA repair genes are duplicated in protozoan parasites, while there is only one gene in yeast. For example, the HRR machinery includes two *rad51* genes in *P. falciparum*, two *rad54* and *mre11* genes in *E. histolytica* [Lopez-Casamichana et al., 2008], two *rpa1* genes in *T. vaginalis*, and two *sgs1* genes in *G. lamblia* and *P. falciparum*. We also identified two *rad27* genes in *P. falciparum* and *G. lamblia* NHEJ pathway, two *E. histolylica ntg1* and *P. falciparum pcna* genes in the BER pathway, as well as two *msh2* genes for the MMR pathway in *E. histolytica* and *P. falciparum*. But the most duplicated gene was the *rad3* gene from the NER mechanism, since there are three genes in *E. histolytica*, two in *G. lamblia* and six in *T. vaginalis*, whereas *P. falciparum* has only one *rad3* gene, alike yeast. Remarkably, gene duplication is evident for many other genes in *T. vaginalis* and reflexes the massive gene expansion inside the large genome of this pathogen [Hartl & Wirth, 2006]. In yeast, the RAD3 protein is involved in mitotic recombination and spontaneous mutagenesis, becoming essential for cell viability in the absence of DNA injury. Furthermore, this protein participates in the repair of UV-irradiated DNA via NER, and constitutes a subunit of RNA polII initiation factor TFIIH [Moriel-Carretero & Aguilera, 2010]. *S. cerevisiae* RAD3 is related to the *H. sapiens* XPD, also known as ERCC2. Defects in human XPD result in a wide range of diseases, including Xeroderma pigmentosum (XP), Cockayne's syndrome, and Trichothiodystrophy characterized by a wide spectrum of symptoms ranging from cancer

In order to describe the inferred evolutionary relationships among the most abundant duplicated gene found through the analysis of DNA repair machineries from the human pathogens studied here, we have undertaken a phylogenetic analysis of RAD3 helicase orthologues in *S. cerevisiae*, *E. histolytica*, *T. vaginalis*, *G. lamblia* and *P. falciparum*. We evaluated the minimum evolution of RAD3 proteins through the construction of Neighbor-Joining phylogenetic tree using the *MEGA* version 5.05 [Tamura et al., 2011]. The robustness was established by bootstrapping test, involving 500 replications of the data based on the criteria of 50% majority-rule consensus (**Fig. 3**). Two main branches that came from a common ancestor can be observed. On one branch, *T. vaginalis* RAD3 parologues are clustered into two sister proteins pairs (A2E1B9 and A2ELX1, A2E4I6 and A2DDD4), that have each evolved from the same ancestor. Besides, *E. histolytica* C4M6T8 is closer to *T. vaginalis* A2E4I6 and A2DDD4, than to its own paralogues. The other branch supports *T. vaginalis* A2G2G8 that is closely related to yeast and *P. falciparum* RAD3 proteins that came off the same node. Interestingly, these two organisms only have one *rad3* gene. This branch also includes *E. histolytica* C4M8K7 and C4M8Q4 sister proteins pair. Intriguingly, the two *Giardia* RAD3 proteins have emerged from different nodes and appeared to be more related to orthologues from other species than to each other; particularly, the branch supporting *Giardia* A8B495 also includes *Trichomonas* A2E1B9 and A2ELX1, while *Giardia* A8BYS3 is on the other branch, isolated from the other proteins, such as *Trichomonas* A2F1W2, which

susceptibility to neurological and developmental defects [Liu et al., 2008].

suggested that these proteins have evolved early.

endonuclease activity in a *P. falciparum* cell free lysate. Authors provide evidence for the presence of class II, Mg2+–dependent and independent AP endonucleases in the extracts. Moreover, they detected that *Plasmodium* AP endonuclease(s) possessed a 3´ phosphodiesterase activity similar to those described in other class II AP endonucleases Demple et al., 1986. In a related study, it was reported that a *P. falciparum* lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities Haltiwanger et al., 2000. In contrast, DNA repair activities in cell lysates have not been detected in *Entamoeba, Giardia* and *Trichomonas* parasites. These data remark the utility of cell free lysates to understand DNA repair pathways, and pointed out to the urgency to investigate endogenous DNA repair activities using whole cell extracts in parasites where no data is available.
