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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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| Killelea, Tom; Palud, Adeline; Akcha, Farida; Lemor, Mélanie; L'Haridon, Stephane; Godfroy, Anne; Henneke, Ghislaine. |
| 8-oxodeoxyguanosine (8-oxodG), a major oxidised base modification, has been investigated to study its impact on DNA replication in hyperthermophilic Archaea. Here we show that 8-oxodG is formed in the genome of growing cells, with elevated levels following exposure to oxidative stress. Functional characterisation of cell-free extracts and the DNA polymerisation enzymes, PolB, PolD, and the p41/p46 complex, alone or in the presence of accessory factors (PCNA and RPA) indicates that translesion synthesis occurs under replicative conditions. One of the major polymerisation effects was stalling, but each of the individual proteins could insert and extend past 8-oxodG with differing efficiencies. The introduction of RPA and PCNA influenced PolB and PolD in... |
| Tipo: Text |
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| Ano: 2019 |
URL: https://archimer.ifremer.fr/doc/00502/61372/64989.pdf |
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| Ralec, Celine; Henry, Etienne; Lemor, Melanie; Killelea, Tom; Henneke, Ghislaine. |
| Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequences of replacing Mg2+ by Ca2+ on base pairing at the polymerase active site as well as the editing of terminal nucleotides at the exonuclease active site of the archaeal Pyrococcus abyssi DNA Pol (PabPolB) are characterized and compared to other (families B, A, Y, X, D) DNA Pols. Based on primer extension assays, steady-state kinetics and ion-chased experiments, we... |
| Tipo: Text |
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| Ano: 2017 |
URL: http://archimer.ifremer.fr/doc/00405/51663/52210.pdf |
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