The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.

En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/00002/11293/7879.pdfInternational audienceA glycine-rich motif described as being involved in human polymerase delta proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D

Novel inhibition of archaeal family-D DNA polymerase by uracil.

International audienceArchaeal family-D DNA polymerase is inhibited by the presence of uracil in DNA template strands. When the enzyme encounters uracil, following three parameters change: DNA binding increases roughly 2-fold, the rate of polymerization slows by a factor of ≈ 5 and 3'-5' proof-reading exonuclease activity is stimulated by a factor of ≈ 2. Together these changes result in a significant decrease in polymerization activity and a reduction in net DNA synthesis. Pol D appears to interact with template strand uracil irrespective of its distance ahead of the replication fork. Polymerization does not stop at a defined location relative to uracil, rather a general decrease in DNA synthesis is observed. 'Trans' inhibition, the slowing of Pol D by uracil on a DNA strand not being replicated is also observed. It is proposed that Pol D is able to interact with uracil by looping out the single-stranded template, allowing simultaneous contact of both the base and the primer-template junction to give a polymerase-DNA complex with diminished extension ability

Binding to PCNA in Euryarchaeal DNA Replication requires two PIP motifs for DNA polymerase D and one PIP motif for DNA polymerase B

En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/2009/publication-7317.pdfInternational audienceReplicative DNA polymerases possess a canonical C-terminal proliferating cell nuclear antigen (PCNA)-binding motif termed the PCNA-interacting protein (PIP) box. We investigated the role of the PIP box on the functional interactions of the two DNA polymerases, PabPol B (family B) and PabPol D (family D), from the hyperthermophilic euryarchaeon Pyrococcus abyssi, with its cognate PCNA. The PIP box was essential for interactions of PabPol B with PCNA, as shown by surface plasmon resonance and primer extension studies. In contrast, binding of PabPol D to PCNA was affected only partially by removing the PIP motif. We identified a second palindromic PIP box motif at the N-terminus of the large subunit of PabPol D that was required for the interactions of PabPol D with PCNA. Thus, two PIP motifs were needed for PabPol D for binding to PabPCNA. Moreover, the C-terminus of PabPCNA was essential for stimulation of PabPol D activity but not for stimulation of PabPol B activity. Neither DNA polymerase interacted with the PabPCNA interdomain connecting loop. Our data suggest that distinct processes are involved in PabPol D and PabPol B binding to PCNA, raising the possibility that Archaea require two mechanisms for recruiting replicative DNA polymerases at the replication fork

In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways.

International audienceUsing 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 polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments

Real-Time Fluorescence Assays to Monitor DNA Polymerase Activities

International audienceSometimes even within the same subfamily, the mechanistic properties of two helicases can be very different. S. cerevisiae Upf1 (yUpf1) and human Smubp-2 are two helicases from Upf1-like family that belong to SF1B. In this work we show that despite having a similar helicase core, the helicase domain (HD) of these two enzymes work in a very different manner. yUpf1-HD has a strong binding affinity to ssDNA and a very high processivity, whereas Smubp-2-HD has week binding to ssDNA and almost unrecognizable processivity. Helicase cores of Smubp2 and Upf1 have similar folds: they contain two RecA-like domains (1A and 2A) and two sub-domains, 1B and 1C, inserted in domain 1A. Rec-A like domains are believed to act as a motor in a helicase. Hence it is puzzling why the two helicases behave so differently despite having highly conserved domains 1A and 2A. We try to identify the role of sub-domains 1B and 1C in this respect. We have utilized a magnetic tweezers based single molecule assay, acting in parallel on tens of molecules at the same time, which allows detecting the unwinding of DNA hairpin substrates. Using this system, we have studied various chimeras of yUpf1-HD and Smubp-2-HD. We show that domains 1B and 1C play an equally important role in processivity of these helicases

Caractérisation des ADN polymérases de Pyrococcus abyssi en présence de matrices ADN endommagées

Les archaea hyperthermophiles colonisent des biotopes où sévissent des conditions extrêmes de température, de pH, des radiations ionisantes, propices à l apparition de lésions dans l ADN. L euryarchaea hyperthermophile Pyrococcus abyssi (Pab) a été utilisée ici comme modèle pour étudier l impact de ces lésions sur la maintenance génomique. La lére partie, consacrée à la détection des lésions dans le génome de Pab, a montré pour la 1ère fois que Pab possède un taux plus élevé de sites AP et de lésions 8-oxo-dG que E.coli. Suite à un stress oxydatif en bioréacteur gas-lift, l augmentation du taux de 8-oxo-dG observée est corrélée à une diminution importante de la survie cellulaire, soulignant l effet génotoxique du stress chez Pab. Le 2nd objectif a été d élucider le comportement des ADN polymérases réplicatives de Pab, PabpolB (Famille B) et PabpolD (Famille D), en présence d ADN endommagé. Bien qu elles réussissent à franchir les lésions 8-oxo-dG et 8-oxo-dA, elles sont bloquées par un site AP. L étude des paramètres cinétiques d incorporation a démontré la haute fidélité des Pabpols en présence d ADN non endommagé. Néanmoins, elles sont sensibles à la présence d un site AF et favorisent l incorporation d un dAMP face à ce dommage. Leur fonction exonucléasique joue alors un rôle important dans la protection contre la mutagénèse en agissant comme une barrière cinétique à l incorporation. L insertion du dAMP est aussi privilégiée en face de la lésion 8-oxo-dG par les 2 Pabpols. Face à la lésion 8-oxo-dA, seule PabpolD incorpore majoritairement un dAMP et un dTMP. La 3ème partie biotechnologique a souligné la capacité des Pabpols à amplifier de l ADN endommagé par PCR.Hyperthermophilic archaea cope with harsh conditions such as high temperatures, pH shifts and ionising radiations. Such environments favour the apparition of DNA lesions, Here, Pyrococcus abyssi (Pab) was used as an attractive model to analyse the impact of DNA lesions onto the maintenance of genome integrity. The 1st part aimed at detecting DNA damages in the genome of Pab and showed that endogenous AP sites and the oxidized DNA base 8-oxo-dG persist at a higher level in Pab genome compared with E.coli. Under oxydative stress conditions performed in a gas-lift bioreactor, the 8-oxo-dG increasing rate is correlated to the important cell survival diminution, assessing the genotoxic effects of this stress on Pab. The 2nd objective was to unravel the behaviour of the replicative DNA polymerases from Pab, PabpolB (Familly B) and PabpolD (Familly D). in presence of damaged DNA. Both Pabpols are able to bypass 8- oxo-dG and 8-oxo-dA whereas they are blocked by the AP site. Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site albeit inefficiently. Their exonuclease function seemed to endow with an important role in the protection against mutagenesis since it acts as a kinetic barrier, preventing misincorporation. The incorporation of the dAMP is also favoured in front of 8-oxo-dG by both Pabpols. However, only Pabpol inserts a dAMP or a dTMP opposite 8-oxo-dA. The biotechnological 3rd part showed that both Pabpols are able to amplify damaged DNA by PCR.BREST-BU Droit-Sciences-Sports (290192103) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi

International audienceReplication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab) slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+) and exonuclease deficient (exo-) forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity P furiosus (Pfu) DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences

Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

International audienceArchaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi b-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs

Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII▿

Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of Pyrococcus abyssi showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme. PabRNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA template. On the other hand, a 5′ RNA flap Okazaki fragment intermediate impaired PabRNase HII endonuclease activity. Furthermore, introduction of mismatches into the RNA portion near the RNA-DNA junction decreased both the specificity and the efficiency of cleavage by PabRNase HII. Additionally, PabRNase HII could cleave a single ribonucleotide embedded in a double-stranded DNA. Our data revealed PabRNase HII as a dual-function enzyme likely required for the completion of DNA replication and DNA repair