Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p>In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. <it>RAD52 </it>epistasis group genes involved in recombinational DNA repair, including <it>mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 </it>and <it>rad59 </it>genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in <it>Entamoeba histolytica </it>the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.</p> <p>Results</p> <p>In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote <it>E. histolytica </it>using UV-C (150 J/m²) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In <it>E. histolytica </it>genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the <it>E. histolytica </it>RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear <it>foci</it>-like structures in <it>E. histolytica </it>trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands <it>in vitro</it>.</p> <p>Conclusion</p> <p><it>E. histolytica </it>genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear <it>foci</it>-like structures. Functional assays confirmed that EhRAD51 is a <it>bonafide </it>recombinase. These data provided the first insights about the potential roles of the <it>E. histolytica </it>RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.</p

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in -6

Ee probe. Lanes 2 to 4, ssDNA incubated with increasing amounts of rEhRAD51 (2.5, 5 and 7.5 μg, respectively); lanes 5 to 7, ssDNA incubated with increasing concentrations of mock purified fraction (2.5, 5 and 7.5 μg) as control. Protein-DNA complexes (Cto C) are shown by arrowheads. . Partially purified rEhRAD51 was incubated with [α-P]dATP labeled dsDNA and interactions were resolved through PAGE. Lane 1, free probe. Lanes 2 to 4, dsDNA incubated with increasing amounts of rEhRAD51 (2.5, 5 and 7.5 μg, respectively); lanes 5 to 7, dsDNA incubated with increasing concentrations of mock purified fraction elution fraction (2.5, 5 and 7.5 μg) as control. Protein-DNA complexes (Cto C) are shown by arrowheads. . D-loop reactions containing 10,000 cpm of [γ-P]dATP-labeled oligonucleotide, circular dsDNA and 0, 2.5, 5 and 7.5 μg of partially-purified rEhRAD51 (lanes 1 to 4) were incubated at 37°C for 30 min with 2 mM of ATP. Negative controls were performed without homologous dsDNA (lane 5) and with heterologous dsDNA oligonucleotide instead of homologous dsDNA (lane 6), both of them using 7.5 μg of EhRAD51 elution fraction. Reaction products were analyzed by agarose gel electrophoresis, transferred to nylon membranes and visualized through a Phosphor Imager. . Densitometric analysis of D-loop products obtained in C. Results are representative of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in "</p><p>http://www.biomedcentral.com/1471-2199/9/35</p><p>BMC Molecular Biology 2008;9():35-35.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2324109.</p><p></p

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in -0

Non-irradiated (No UV-C) and irradiated (UV-C) trophozoites harvested at different times (30 min, 3, 6 and 12 h). Upper panels, histograms show the DNA fragmentation percentage in fluorescence positive cells. The abscissa indicates fluorescence of propidium iodide (PI), and the ordinate indicates fluorescence of Alexa 488-labeled 3' ends of DNA. The number inside each histogram denotes the percentage of fluorescence positive cells above the cut-off line. Lower panels, PI-staining cells were checked in the epifluorescence microscope to confirming the absence of cytoplasmic stain. PI, propidum iodide, N, Nomanski optics. . Neutral comet assays of non-irradiated (No UV-C) and irradiated (UV-C) trophozoites harvested at different times (30 min, 3, 6 and 12 h). Electrophoretic migration of DNA was from left (anode) to right (cathode).<p><b>Copyright information:</b></p><p>Taken from "Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in "</p><p>http://www.biomedcentral.com/1471-2199/9/35</p><p>BMC Molecular Biology 2008;9():35-35.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2324109.</p><p></p

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in -2

D trophozoites harvested at different times (UV-C; lane 2, 0.5 h; lane 3, 3 h and lane 4,12 h). Arrowheads denote the length (bp) of each expected amplified internal fragment, as described in Table 2. . Densitometric analyses of RT-PCR products in A. Pixels corresponding to the rRNA product were taken as 100% in each lane. Data are the mean of three independent assays.<p><b>Copyright information:</b></p><p>Taken from "Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in "</p><p>http://www.biomedcentral.com/1471-2199/9/35</p><p>BMC Molecular Biology 2008;9():35-35.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2324109.</p><p></p

Protein Kinases and Transcription Factors Activation in Response to UV-Radiation of Skin: Implications for Carcinogenesis

Solar ultraviolet (UV) radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer

Human Papillomavirus Coinfection in the Cervical Intraepithelial Lesions and Cancer of Mexican Patients

According to their oncogenic properties, Human Papillomaviruses (HPVs) are classified into two types: Low-Risk (LR-HPVs) and High-Risk Human Papillomaviruses (HR-HPVs). The immune system naturally controls the majority of HPV infections; however, when the HR-HPV infection is persistent, the risk of developing cervical cancer increases. Previous studies indicate that multiple-infection or coinfection with HR-HPV occurs frequently and can potentiate the development of cervical lesions. This study aimed to establish the HPV coinfection rate in squamous intraepithelial lesions from Mexican patients. For HPV detection, we performed PCR on 55 cervical lesions diagnosed by colposcopy. We detected the presence of HPV infection in 87.27% (48/55) of the lesions; interestingly, HPV coinfection was observed in 70.83% (34/48) of these samples. We also evaluated HPV infection in adjacent areas without morphological changes from 25 samples. The results showed that 80% (20/25) of these were HPV-positive and, curiously, all presented HPV-16 infection. In conclusion, our results revealed a high prevalence of HPV coinfection in cervical lesions in Mexican patients, and these results contribute to future research focused on the role that HPV coinfection plays in the development of cervical cancer

Protein Phosphorylation in Serine Residues Correlates with Progression from Precancerous Lesions to Cervical Cancer in Mexican Patients

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions