Identification of insertion hot spots for non-LTR retrotransposons: computational and biochemical application to Entamoeba histolytica

Mandal, Prabhat Kumar; Rawal, Kamal; Ramaswamy, Ramakrishna; Bhattacharya, Alok; Bhattacharya, Sudha

doi:10.1093/nar/gkl710

Cited by 17 publications

(29 citation statements)

References 39 publications

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“…Although the enzyme is not strictly sequence specific, it nicks preferentially at certain hot-spots. The consensus hot-spot sequence is 5¢-GCATT-3¢, with nicks occurring between A-T and T-T [10]. Here we report some of the salient enzymatic features of the EhLINE1-EN, and discuss the results with respect to its role in retrotransposition.…”

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confidence: 84%

Characterization of the restriction enzyme‐like endonuclease encoded by the Entamoeba histolytica non‐long terminal repeat retrotransposon EhLINE1

et al. 2009

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The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non‐long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1‐encoded endonuclease catalyzed reaction, determined under steady‐state and single‐turnover conditions, revealed a significant burst phase followed by a slower steady‐state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the Km was 2.6 × 10−8 m and the kcat was 1.6 × 10−2 sec−1. For linear E. histolytica DNA substrate the Km and kcat values were 1.3 × 10−8 m and 2.2 × 10−4 sec−1 respectively. Single‐turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg2+ was required for activity, 60% activity was seen with Mn2+ and none with other divalent metal ions. Substitution of PDX12‐14D (a metal‐binding motif) with PAX12‐14D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1‐encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.

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Characterization of the restriction enzyme‐like endonuclease encoded by the Entamoeba histolytica non‐long terminal repeat retrotransposon EhLINE1

et al. 2009

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“…A tri-nucleotide model based on DNase-I cutting frequencies predicts that DNase I binds and cuts DNA that is bent toward major groove 5 , 6 , 8 …”

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confidence: 99%

“…The genomic hotspots are identified by DNA structure and, endonuclease (EN) nicking to that DNA sequence 5 . These insertion hot spots are characterized by presence of sequence motifs and unique patterns 6 .…”

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Identification and characterization of MGEs and their insertion sites in thegorillagenome

Rawal

Dorji

Kumar

et al. 2013

Mobile Genetic Elements

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Recently published gorilla genome has offered an opportunity to study human evolution through variety of approaches. Mobile genetic elements (MGEs) insert non randomly in genome through mechanisms such as retrotransposition and may cause gene inactivation, transduction, regulation of gene expression and genome expansion. Here we report that majority of gorilla genome is occupied with MGEs (> 36%) with presence of LTRs and Non-LTRs such as Alus and L1s. Other types of MGEs such as MIRs, retrovirus like elements ERVs and DNA transposons are also found using repeatmasker and ELAN pipeline. The distribution is similar to Humans and Macaca genome. Using DNA Scanner we also scanned preinsertion loci for number of different properties such as DNA denaturation, energy measures, potential for protein interactions and sequence based features. We also predicted preinsertion loci with > 70% accuracy using a machine learning tool called insertion site finder (ISF) based upon support vector machines.

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Section: Properties Of the Ehline1-endonucleasementioning

confidence: 99%

“…The sequence preference of the EN was determined in vitro with a variety of mutated substrates. It was possible to assign a consensus sequence, 5'-GCATT-3', which was efficiently nicked between A-T and T-T (Mandal et al, 2006).Studies on the kinetics of EhLINE1 ENcatalyzed reaction, determined under steady-state and single turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction (Yadav et al, 2009). Like restriction endonucleases, this enzyme displayed a low K m , suggesting high affinity for DNA.…”

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Circular Noncoding Ribosomal Transcripts and Recombination of Retrotransposons: Two Charming Secrets Revealed by a Less-Explored Human Parasite

Bhattacharya¹

2015

Proceedings of the Indian National Science Academy

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Parasites, through their diverse cellular metabolic pathways, help us to appreciate the different directions in which evolution works, while maintaining a unified theme. To illustrate this I describe our recent observations with two basic processes, namely regulation of ribosomal RNA transcription, and the biology of retrotransposition in Entamoeba histolytica, a highly prevalent protozoan parasite that causes amebiasis.Ribosomal RNA synthesis is generally tightly regulated in response to growth rate such that cells subjected to growth stress shut down their rRNA transcription. In E. histolytica we observed that upon growth stress, rRNA synthesis did not shut down. Instead, unprocessed pre-rRNA accumulated to high levels along with a novel class of circular RNAs derived from the 5'-external transcribed spacer (etsRNA). The etsRNA can self circularize in vitro, a property not previously known in spacer RNAs. We hypothesize that circular etsRNAs would escape exonucleolytic decay and inhibit pre-rRNA processing, possibly by titrating away the processing factors which normally bind to them.In the study on retrotransposition of non-long terminal repeat retrotransposons (EhLINEs/SINEs), we successfully mobilized EhSINE in a cell-line made retrotransposition-competent by transfection with multiple constructs to express the polypeptides required for retrotransposition, a first for any protozoan parasite. While tracking retrotransposition of a marked SINE copy we found that the newly retrotransposed SINEs had undergone high-frequency recombination, presumably due to the known ability of reverse transcriptase to perform template jumping. Such recombination has not been reported for retrotransposons, and may be important in generating sequence polymorphism.Key Words: Entamoeba histolytica; Circular Non Coding RNA; Ribosomal RNA Spacer; Ehsine; Non LTR Retrotransposon; SINE Recombination IntroductionEntamoeba histolytica, a protozoan parasite, is classically defined as the causative agent of the disease Amoebiasis. It resides in the human colon along with a number of nonpathogenic species of Entamoeba. The estimates available world-wide state that 500 million persons are infected with Entamoeba spp., with about 50 million cases of invasive amoebiasis per year; resulting in about 100,000 deaths (WHO/PAHO/UNESCO report, 1997). The parasite is directly transmitted through the faecal-oral route without the need of a vector system. It therefore flourishes where humans live in crowded conditions with poor sanitation. It continues to be a major public 432 Sudha Bhattacharya health problem in India due to recurrent infections caused by the high parasite load in our environment, and lack of effective immunity in infected individuals. The morbidity caused by the intestinal form of the disease results in great economic loss, although mortality is reduced, thanks to the freely-available and over-used anti-amoebic drug, metronidazole (Upcroft and Upcroft, 2001). The parasite also invades the liver and other organs, causing abscesses ...

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Identification of insertion hot spots for non-LTR retrotransposons: computational and biochemical application to Entamoeba histolytica

Cited by 17 publications

References 39 publications

Characterization of the restriction enzyme‐like endonuclease encoded by the Entamoeba histolytica non‐long terminal repeat retrotransposon EhLINE1

Characterization of the restriction enzyme‐like endonuclease encoded by the Entamoeba histolytica non‐long terminal repeat retrotransposon EhLINE1

Identification and characterization of MGEs and their insertion sites in thegorillagenome

Circular Noncoding Ribosomal Transcripts and Recombination of Retrotransposons: Two Charming Secrets Revealed by a Less-Explored Human Parasite

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