**1. Introduction**

36 Will-be-set-by-IN-TECH

368 Selected Topics in DNA Repair

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Numerous external and internal DNA damaging agents can affect genetic material to produce single-strand breaks (SSB), double strand breaks (DSB), inter- and intra-strand cross-links in the form of cyclobutane pyrimidine dimers and (6-4)-photoproducts, oxidation and alkylation of bases, or formation of bulky chemical adducts. Cells possess several biological processes that act in a coordinated way to supervise DNA molecules and properly repair DNA lesions to minimize genetic information loss. This DNA repair system, which has been conserved throughout eukaryotes and prokaryotes evolution, includes various pathways that can be classified according to the type of DNA lesion they can restore: i) DSB, the most detrimental lesions of DNA, can be repaired by homologous recombination (HRR) and non-homologous end joining (NHEJ) pathways [Fleck & Nielsen, 2004]; ii) aberrant bases or nucleotides from a single strand DNA can be repaired by base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways using the complementary strand as template for DNA synthesis. BER mainly restores non-bulky lesions that result from bases alkylation, oxidation or deamination [Krokan et al., 1997]. The main task of NER pathway, which consists in two subpathways: global genome repair (GGR) to remove damage in the overall genome and transcription-coupled repair (TCR) to specifically repair the transcribed strand of active genes, is to eliminate photoproducts produced by ultraviolet (UV) light and other bulky lesions, such as inter- and intra-strand crosslinks [Prakash &Prakash, 2000]. MMR allows the removal of base mismatches and small insertion/deletion loops (IDL) that are formed during the replication process [Marti et al., 2002].

The genome of protozoan parasites is continuously subjected to the effects of antiparasitic drugs and host immune system attacks, which can affect its stability and therefore parasite survival. Thus, efficient DNA maintenance mechanisms are necessary to detect and accurately repair damaged nucleotides. The fully sequenced genome of the four major human pathogens described here provides new insights into parasite biology, including molecular features of

DNA Repair in Pathogenic Eukaryotic Cells:

*histolytica* [Lopez-Casamichana et al., 2008].

was too divergent to be detected by our *in silico* strategy.

Insights from Comparative Genomics of Parasitic Protozoan 371

*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

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.* 

*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

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

organisms use highly divergent KU proteins to perform the NHEJ pathway.

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.
