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| Lemor, Melanie; Kong, Ziqing; Henry, Etienne; Brizard, Raphael; Laurent, Sebastien; Bosse, Audrey; Henneke, Ghislaine. |
| Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a... |
| Tipo: Text |
Palavras-chave: Archaea; DNA replication and repair; DNA polymerase; Nucleotide pool; Translesion synthesis. |
| Ano: 2018 |
URL: https://archimer.ifremer.fr/doc/00464/57603/59796.pdf |
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| Henneke, Ghislaine. |
| Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi. I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5'-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5'-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by... |
| Tipo: Text |
Palavras-chave: Archaea; DNA ligase; DNA polymerase; Nuclease; Okazaki fragment. |
| Ano: 2012 |
URL: http://archimer.ifremer.fr/doc/00107/21841/20923.pdf |
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| Zhang,Junhong; Li,Guohui; Chen,Huiqing; Li,Xiaogang; Lv,Meng; Chen,Keping; Yao,Qin. |
| BmPLV-Z is the abbreviation for Bombyx mori parvo-like virus (China isolate). This is a novel virus with two single-stranded linear DNA molecules, viz., VD1 (6543 bp) and VD2 (6022 bp), which are encapsidated respectively into separate virions. Analysis of the deduced amino acid sequence of VD1-ORF4 indicated the existence of a putative DNA-polymerase with exonuclease activity, possibly involved in the replication of BmPLV-Z. In the present study, a recombinant baculovirus was constructed to express the full length of the protein encoded by the VD1-ORF4 gene (3318 bp). In addition, a 2163-bp fragment amplified from the very same gene was cloned into prokaryotic expression vector pET-30a and expressed in E.coli Rosetta 2 (DE3) pLysS. The expressed fusion... |
| Tipo: Info:eu-repo/semantics/article |
Palavras-chave: Bombyx mori parvo-like virus; DNA polymerase; Recombinant protein; Sf-9. |
| Ano: 2010 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572010000400021 |
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| Killelea, Tom; Ralec, Celine; Bosse, Audrey; Henneke, Ghislaine. |
| DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides,... |
| Tipo: Text |
Palavras-chave: DNA polymerase; Archaea; Family D; PCR; Pyrococcus. |
| Ano: 2014 |
URL: http://archimer.ifremer.fr/doc/00193/30450/28871.pdf |
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| Hogrel, Gaelle; Lu, Yang; Alexandre, Nicolas; Bossé, Audrey; Dulermo, Remi; Ishino, Sonoko; Ishino, Yoshizumi; Flament, Didier. |
| Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show... |
| Tipo: Text |
Palavras-chave: Homologous recombination; Archaea; DNA polymerase; Recombinase; DNA repair. |
| Ano: 2020 |
URL: https://archimer.ifremer.fr/doc/00641/75274/75539.pdf |
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| Henneke, Ghislaine; Flament, Didier; Hubscher, Ulrich; Querellou, Joel; Raffin, Jean-paul. |
| DNA polymerases carry out DNA synthesis during DNA replication, DNA recombination and DNA repair. During the past five years, the number of DNA polymerases in both eukarya and bacteria has increased to at least 19 and multiple biological roles have been assigned to many DNA polymerases. Archaea, the third domain of life, on the other hand, have only a subset of the eukaryotic-like DNA polymerases. The diversity among the archaeal DNA polymerases poses the intriguing question of their functional tasks. Here, we focus on the two identified DNA polymerases, the family B DNA polymerase B (PabpolB) and the family D DNA polymerase D (PabpolD) from the hyperthermophilic euryarchaeota Pyrococcus abyssi. Our data can be summarized as follows: (i) both Pabpols are... |
| Tipo: Text |
Palavras-chave: DNA polymerase; Strand displacement; Gap filling; Euryarchaea; DNA replication. |
| Ano: 2005 |
URL: http://archimer.ifremer.fr/doc/2005/publication-423.pdf |
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