Role of ATP hydrolysis in the DNA translocase activity of the bovine papillomavirus (BPV-1) E1 helicase

The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal ∼300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects ∼30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non–hydrolysable ATP analogue AMP–PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T(1/2) > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T(1/2) ∼60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein–protein interactions switch from low to high affinity. This reciprocal change in protein–protein and protein–DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds

Common determinants in DNA melting and helicase-catalysed DNA unwinding by papillomavirus replication protein E1

E1 and T-antigen of the tumour viruses bovine papillomavirus (BPV-1) and Simian virus 40 (SV40) are the initiator proteins that recognize and melt their respective origins of replication in the initial phase of DNA replication. These proteins then assemble into processive hexameric helicases upon the single-stranded DNA that they create. In T-antigen, a characteristic loop and hairpin structure (the pre-sensor 1β hairpin, PS1βH) project into a central cavity generated by protein hexamerization. This channel undergoes large ATP-dependent conformational changes, and the loop/PS1βH is proposed to form a DNA binding site critical for helicase activity. Here, we show that conserved residues in BPV E1 that probably form a similar loop/hairpin structure are required for helicase activity and also origin (ori) DNA melting. We propose that DNA melting requires the cooperation of the E1 helicase domain (E1HD) and the origin binding domain (OBD) tethered to DNA. One possible mechanism is that with the DNA locked in the loop/PS1βH DNA binding site, ATP-dependent conformational changes draw the DNA inwards in a twisting motion to promote unwinding

Caractérisation d'une RNase A-like spécifique de la région proximale de l'épididyme : le Train A

*INRA, PRC, 37380 Nouzilly Diffusion du document : INRA, PRC, 37380 Nouzilly Diplôme : Dr. d'Universit

Caractérisation d'une RNase A-like spécifique de la région proximale de l'épididyme (le Train A)

La maturation des spermatozoïdes dans l'épididyme est due en grande partie à l'interaction avec les protéines secrétées dans la lumière du tube par l'épithélium . Parmi ces protéines, le Train A représente 89% des sécrétions de la région antérieure(zone 0)chez le verrat . Le Train A, avec des isoformes de 26 à 33 kDa et de pI de 5 à 8,5, est le premier membre d'une nouvelle famille de RNase A-like(32% d'identité avec les RNases A)caractérisée par l'absence de la triade catalytique classique et la présence d'un bras N-terminal de 10 kDa en amont du domaine homologue aux RNases A, sans activité RNase . Ses ARN, exprimés spécifiquement dans la région 0 de l'épididyme, sont présents chez toutes les espèces étudiées . Il ne se fixe pas aux spermatozoïdes mais est réabsorbé rapidement et spécifiquement par ce même épithélium pour peut-être exercer une action de type autocrine . L'expression de la protéine et des ARN est simultanée, dès la puberté, et régulée par des facteurs testiculaires.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Identification, function and regulation of Train A (RNase 10): a new epididymal-specific RNase-A like protein

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Prostaglandin D2 synthase secreted in the caput epididymidis displays spatial and temporal delay between messenger RNA and protein expression during postnatal development

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Epididymal cell secretory activities and the role of proteins in boar sperm maturation

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Ilf3 and NF90 functions in RNA biology

International audienceDouble-stranded RNA-binding proteins (DRBPs) are known to regulate many processes of RNA metabolism due, among others, to the presence of double-stranded RNA (dsRNA)-binding motifs (dsRBMs). Among these DRBPs, Interleukin enhancer-binding factor 3 (Ilf3) and Nuclear Factor 90 (NF90) are two ubiquitous proteins generated by mutually exclusive and alternative splicings of the Ilf3 gene. They share common N-terminal and central sequences but display specific C-terminal regions. They present a large heterogeneity generated by several post-transcriptional and post-translational modifications involved in their subcellular localization and biological functions. While Ilf3 and NF90 were first identified as activators of gene expression, they are also implicated in cellular processes unrelated to RNA metabolism such as regulation of the cell cycle or of enzymatic activites. The implication of Ilf3 and NF90 in RNA biology will be discussed with a focus on eukaryote transcription and translation regulation, on viral replication and translation as well as on noncoding RNA field. WIREs RNA 2015, 6:243-256. doi: 10.1002/wrna.1270 For further resources related to this article, please visit the . Conflict of interest: The authors have declared no conflicts of interest for this article

Binding of E1HD and PS1βH and hydrophobic loop mutants to ssDNA, without and with ATP/Mg (indicated simply as ATP), top and bottom panel, respectively

<p><b>Copyright information:</b></p><p>Taken from "Common determinants in DNA melting and helicase-catalysed DNA unwinding by papillomavirus replication protein E1"</p><p>Nucleic Acids Research 2006;34(10):3008-3019.</p><p>Published online 31 May 2006</p><p>PMCID:PMC1474052.</p><p>© 2006 The Author(s)</p> The ssDNA substrate was a 30mer chosen at random, the protein titration series is 250, 1000 and 2000 nM. () ssDNA binding of E1HD and PS1βH mutants as indicated above the lanes. Wild-type is on the left, lanes 2–4; lane 1 is free probe (ssDNA). The ssDNA binding activity of wild-type was enhanced in the presence of ATP (lanes 2–4 lower panel), and R505E recovered partial binding activity. K506A was marked by the appearance of a complex with aberrant mobility (lane 13). Without ATP, H507 substitutions to Leu and Ala incrementally abolished ssDNA binding (lanes 14–19), while the H507F substitution (lanes 20–22) improved activity compared with wild-type. With ATP, ssDNA binding of H507L and H507A improved but was indicative of complex instability. () ssDNA binding of E1HD and hydrophobic loop mutants as indicated above the lanes. Without ATP, DNA binding was practically abolished for all mutants except the conservative substitution F464W. With ATP, below, activity was enhanced for all mutants but most noticeably the conservative substitutions F464W and W465F

The epididymal proteome

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