Phylogenetic Distribution and Evolutionary History of Bacterial DEAD-Box Proteins

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation

Connections : safe spaces for women and youth in Latin America and The Caribbean

RESUMEN: Este libro se puede leer en muchos niveles. Uno de ellos puede no ser muy obvio para aquellos que están acostumbrados a leer sobre violencia e inseguridad en América Latina. Es el nivel que le da a este libro un estatus de originalidad y una contribución que va más allá de la región: el ser una forma de conocimiento destinada no solo a interpretar el mundo, sino a cambiarlo […], visibiliza la importancia de un proceso de investigación ajustado al tipo de conocimiento que produce. Aquí se conectan el proceso y el resultado, lo que debería propiciar un debate más amplio con respecto a cómo y qué sabemos de la naturaleza de la violencia y la agencia social para reducirla […]. Esta visión es particularmente relevante en contextos donde el Estado reproduce la violencia, con terribles impactos, en especial en periferias excluidas. […] El proceso de investigación abordado en este libro transgredió muchas fronteras. Hubo fronteras entre países, barreras lingüísticas, fronteras en torno a la educación, el conocimiento y la experiencia, y entre etnias, géneros y generaciones. […] este proceso reunió a académicos, activistas y líderes comunitarios de cinco países de América Latina y uno del Caribe, incluyendo comunidades indígenas en México y Guatemala […]. La violencia está en el tiempo y en el espacio y se reproduce entre las generaciones en diversos espacios de socialización. Este proceso de investigación que trasciende las fronteras, plantea una discusión que atraviesa los diferentes casos sobre cómo los déficits y las desigualdades materiales, las violencias estatales en nombre de la ‘seguridad’, las especificidades culturales, de género y generacionales de la experiencia y la comprensión de la violencia, así como las diversas formas de criminalidad, se cruzan y se reproducen a través del tiempo y el espacio. Jenny Pearce, investigadora y profesora en el Latin American and Caribbean Centre (LACC) de la London School of Economics and Political ScienceABSTRACT: This book can be read on many levels. One level may not be so obvious to those who are used to reading about violence and insecurity in Latin America. It is the level which gives this book a claim to true originality and a contribution beyond the region. This contribution is to form of scholarship aimed not only to interpret the world but to change it […], this text visibilizes the significance of the research process to the kind of knowledge that is produced. It connects process and outcome, and this should start a wider debate about how as well as what we know about the nature of violence and the social agency to reduce it […]. This is particularly relevant in contexts where the State reproduces violence, with terrible impacts on the margins. The research process discussed in this book transgressed many boundaries. There were intercountry borders, linguistic barriers, boundaries around education, knowledge and experience and between ethnicities, genders and generations. […] the research process brought together scholars and community activists and actors from five Latin American and one Caribbean country. And within Latin America there were indigenous communities in Mexico and Guatemala who participated […]. Violence is located in time and space. It is reproduced inter-generationally through varied socialisation spaces. The boundary crossing research process, raises cross case discussion about how material deficits and inequalities, state violences in the name of ‘security’, cultural, gender and generational specificities of experience and understanding of violence, and varied forms of criminality, intersect and reproduce through time and space. Professor Jenny Pearce. Latin American and Caribbean Centre (LACC), London School of Economics and Political Scienc

A transposon-derived DNA polymerase from Entamoeba histolytica displays intrinsic strand displacement, processivity and lesion bypass.

Entamoeba histolytica encodes four family B2 DNA polymerases that vary in amino acid length from 813 to 1279. These DNA polymerases contain a N-terminal domain with no homology to other proteins and a C-terminal domain with high amino acid identity to archetypical family B2 DNA polymerases. A phylogenetic analysis indicates that these family B2 DNA polymerases are grouped with DNA polymerases from transposable elements dubbed Polintons or Mavericks. In this work, we report the cloning and biochemical characterization of the smallest family B2 DNA polymerase from E. histolytica. To facilitate its characterization we subcloned its 660 amino acids C-terminal region that comprises the complete exonuclease and DNA polymerization domains, dubbed throughout this work as EhDNApolB2. We found that EhDNApolB2 displays remarkable strand displacement, processivity and efficiently bypasses the DNA lesions: 8-oxo guanosine and abasic site.Family B2 DNA polymerases from T. vaginalis, G. lambia and E. histolytica contain a Terminal Region Protein 2 (TPR2) motif twice the length of the TPR2 from φ29 DNA polymerase. Deletion studies demonstrate that as in φ29 DNA polymerase, the TPR2 motif of EhDNApolB2 is solely responsible of strand displacement and processivity. Interestingly the TPR2 of EhDNApolB2 is also responsible for efficient abasic site bypass. These data suggests that the 21 extra amino acids of the TPR2 motif may shape the active site of EhDNApolB2 to efficiently incorporate and extended opposite an abasic site. Herein we demonstrate that an open reading frame derived from Politons-Mavericks in parasitic protozoa encode a functional enzyme and our findings support the notion that the introduction of novel motifs in DNA polymerases can confer specialized properties to a conserved scaffold

TPR2 is required for efficient strand-displacement.

<p>Strand displacement was assessed using a set of 3 oligonucleotides with gaps of 1, 3 and 6 nt respectively. After incubation at the indicated times the reaction mixtures were run on a 18% denaturing polyacrylamide gel. Reactions were carried out in 20 µl as described in material in methods (<b>A</b>) <b>Strand-displacement activity of EhDNApolB2</b>. Primer extension (lanes 2 to 5), primer extension with 1 nt gap (lanes 6 to 9), primer extension with 3 nt gap (lanes 10 to 13), primer extension with 6 nt gap (14 to 17). (<b>B</b>) <b>Strand-displacement activity of</b> Δ<b>TPR2</b> Primer extension (lanes 2 to 5), primer extension with 1 nt gap (lanes 6 to 9), primer extension with 3 nt gap (lanes 10 to 13), primer extension with 6 nt gap (14 to 17).</p

Modular organization of family B2 DNA polymerases in <i>E. histolytica</i> and structural model of EhDNApolB2.

<p>(<b>A</b>) <i>E. histolytica</i> contains four family B2 DNA polymerases in its genome. These DNA polymerase present a C-terminal region with conserved exonuclease and polymerase motifs characteristic of a family B2 DNA polymerases (green, blue and red boxes). The N-terminal region, indicated by a thin line, presents no similitude to other proteins and is composed of 180 to 500 amino acids. The shortest DNA polymerase is present at loci EHI_018010 and is dubbed EhDNApolB2 throughout this work (<b>B</b>) Homology structural model of EhDNApolB2. The 3′–5′ exonuclease domain is shown in green and the 5′–3′ polymerization domain is shown in blue. The extended TPR2 motif is shown in red encircling double stranded DNA (yellow colored).</p

ΔTPR2 bypasses 8oxoG, but not an abasic site.

<p>Lesion bypass of EhDNApolB2 (lanes 1 to 14) and ΔTPR2 (lanes 15 to 28). The time course primer extension is described as in material and methods using equal amounts of DNA polymerases and 100 µM dNTPs. After incubation times of 2.5, 5, 10 and 20 minutes the primer extension reactions were stopped and run onto a 15% denaturing polyacrylamide gel.</p

EhDNApolB2 efficiently bypasses 8-oxo guanosine and abasic site lesions.

<p>Denaturing polyacrylamide gel electrophoresis showing translesion bypass of EhDNApolB2 in comparison to undamaged template. Primer extension by EhDNApolB2 using a canonical and damaged substrate. The first nucleotide (canonical or damaged) that serves a template is designated by an <b>X</b>. For the 8-oxoguanosine and abasic site the lesion is located immediately after a primer of 29 nt and for thymine glycol and UV adducts is located immediately after a primer of 16 nt. The label 25, 30 or 17 nt indicate the length of the primer is only one nucleotide opposite the lesion is incorporated. Each reaction was incubated with a 20 nM of EhDNApolB2 and 1 nM of several substrates. Aliquots were taken at 0, 2.5, 5, 10 and 20 minutes. Time course of different substrates were loaded in a 15% denaturing gel. Thymine (lanes 1–5); 8-oxo guanosine (lanes 6–10); abasic site (lanes 11–15); 5 S-6R thymine glycol (lanes 16–20); 5R-6S thymine glycol (lanes 21–24); cis-syn cyclobutane pyrimidine dimer (lanes 25–29); 6-4 photo product (lanes 30–33); The upper arrow depicts the length of the final product substrate and the bottom arrow indicates the used primer.</p

TPR2 is responsible of lesion bypass extension opposite an abasic site.

<p>Lesion bypass of wtEhDNApolB2 (lanes 1 to 5 and 11 to 15) and ΔTPR2 (lanes 6 to 10 and 16 to 20) extending from a primer containing a 3′OH purine or a pyrimidine opposite an abasic site. A primer containing a 3′OH dAMP (lanes 1 to 10) or dCMP (11 to 20) opposite an abasic site was subject to a time course primer extension reaction from 2.5 to 20 minutes using equal amounts of DNA polymerases and 100 µM dNTPs. The reaction products were run onto a 15% denaturing polyacrylamide gel.</p

Fidelity of translesion DNA synthesis of EhDNApolB2.

<p>Translesion bypass fidelity of EhDNApolB 20 nM of exonuclease deficient EhDNApolB were incubated with 1 nM of a set of substrates containing several DNA lesions. The reactions were carried out with four dNTPs or single dNTP addition. The dNTPs were present at a concentration of 15 µM. Samples were taken at 2.5 minutes, stopped with 50 mM EDTA and 90% formamide and run onto a 18% denaturing polyacrylamide gel electrophoresis for their analysis by phosphorimagery. (A) Control thymine (lanes 1 to 5), 8-oxo guanosine (lanes 6 to 10), and abasic site (lanes 11 to 15). (B) 5 S-6R and 5R-6S thymine glycol (lanes 1 to 5 and 6 to 10 respectively). The upper arrow depicts the length of the final product substrate and the bottom arrow indicates the used primer.</p