Resultado final
Reconstitution of Okazaki fragment maturation in P. abyssi.
Prior to the start of REPBIOTECH project, we had detected in P. abyssi short nascent DNA strands with 5' RNA segments whose size (up to 120 base pairs) and structure are similar to eukaryotic Okazaki fragments, indicating that Archaea use RNA primers synthesised by eukaryotic-like primase for replication initiation of leading strands and for synthesis of all Okazaki fragments at the retrograde arm of the replication fork (Myllykallio and Forterre 2000; Myllykallio et al. 2000). The details of how these primers are synthetized and removed during chromosomal DNA replication in Archaea are poorly understood.
The apparent absence of DNA polymerase alpha (required for low fidelity synthesis of DNA primers of eukaryotic Okazaki fragments) had suggested that synthesis of Okazaki fragments is likely to possess unique features in Archaea. At the start of the project, proteins homologous to eukaryotic Dna2 helicase/endonuclease that is required for removal of long RNA/DNA primers from eukaryotic Okazaki fragments had been detected in Archaea. However, during the early phases of our project, biochemical work demonstrated that archaeal Dna2-like proteins are inhibited by 5 RNA segments, strongly suggesting that they are not orthologous to their eukaryotic homologs. Consequently, we have not pursued further the role of archaeal Dna2-like proteins in this project.
To address how RNA primers are removed from archaeal Okazaki fragments, we have cloned and overexpressed a number of P. abyssi proteins with an expected function in maturation of Okazaki fragments (e.g. PCNA replication clamp, Fen1 nuclease, DNA ligase). Our interaction studies using surface plasmon resonance revealed high-affinity interactions between PCNA, Fen1 and DNA ligase I as well as RNaseHII.
We have also identified a novel physical interactions between Fen1-RNase HII, suggesting that activities of RNase HII and Fen1 could be coupled by PCNA during DNA replication and/or repair. Physiological significance of the above interactions detected in vitro were confirmed using pull-down assays. Moreover, physical interactions increased also enzymatic activities of the both Fen1 nuclease and DNA ligase. Altogether these results led to functional model for the Okazaki fragment maturation in Archaea.
- The attempts to solve the structure of the P. abysssi RF-C were unsuccessful due to high instability of the complex. Moreover, the human RF-C structure was solved by other research groups during the course of the project, reducing considerably the priority on this specific objective.
- The structure of the N-terminal part of SsoMCM was determined by molecular replacement using the available homologous structure from Methanobacterium thermoautotrophicum as the search model. A partial solution was obtained for the hexagonal crystal form with the program PHASER. Only three of the four subunits in the asymmetric unit suggested by Matthews crystal packing probability calculation (Kantardjieff and Rupp 2003) could be detected. For the monoclinic crystal form, a complete solution was achieved with three subunits in the AU. Interestingly, by application of the crystallographic symmetry operators to the trimer we were able to build up the ring-shaped hexameric structure.
The DNA complex of the N-terminal part has been solved. The manuscript is under completion.
- DNA nuclease Pab 2263: This protein was identified as an interesting target as a result of the search of novel interacting partners. Pab2263 interacts with PCNA and was recently found to have nuclease activity. The first native protein batch was purified and then crystallized by the sitting drop vapour diffusion method. The crystals belonged to the orthorhombic system and space group C2221 with cell dimensions of a = 78.2 Å, b = 101.1 Å, c = 155.9 Å, a= b =g = 90°. It was found that there were two molecules in the asymmetric unit with a local 2-fold axis at the relative coordinates x,z = (0.0125, 0.2268), which is parallel to crystallographic 21 axis.
An initial diffraction data set was collected at DESY, Hamburg, Germany. Since there is no similar structure available, which could be used in a molecular replacement search, we have chosen the multiple-wavelength anomalous diffraction (MAD) method to solve the structure of Pab2263. A Seleno-methionine substituted derivative protein was expressed and purified. The derivative protein was crystallized in the same space group under similar crystallization conditions as the native protein but with different cell dimensions of a = 81.4 Å, b = 159.8 Å, c = 100.8 Å, a= b = g= 90°. A MAD data set was collected at DESY.
Data analysis revealed that only two of the three methionines were substituted, which gave a relatively weak anomalous diffraction signal in particular for the high resolution X-ray data. Anyway a medium resolution electron density map (at Ü 3.5 Å resolution) could be calculated with the available phase information from the MAD data. The density features of this map suggested that Pab2263 forms a complex ring-like structure.
- DNA polymerase (pol) lambda is homologous to pol beta and has intrinsic polymerase and terminal transferase activities. However, nothing was known about the amino acid residues involved in these activites. In order to precisely define the nucleotide-binding site of human pol lambda, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol beta, which are involved in nucleotide binding. Our analysis demonstrated that pol lambda Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol lambda, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.
- Pol lambda and pol mu are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol mu and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol mu and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.
- Human DNA polymerases (pols) beta and lambda could promote template slippage and generate -1frameshifts on defined heteropolymeric DNA substrates containing a single abasic site. Kinetic data demonstrated that pol ìambda was more efficient than pol beta in catalyzing translesion DNA synthesis past an abasic site, particularly in the presence of low nucleotide concentrations. Moreover, pol lambda was found to generate frameshifts in two ways: first, by using a nucleotide-stabilized primer misalignment mechanism, or second, by promoting primer reannealing using microhomology regions between the terminal primer sequence and the template strand. Our results suggest a molecular mechanism for the observed high in vivo rate of frameshifts generation by pol lambda and highlight the remarkable ability of pol lambda to promote microhomology pairing between two DNA strands, further supporting its proposed role in the nonhomologous end joining process.
- We showed also that DNA polymerase lambda interacts with proliferating cell nuclear antigen (PCNA) in vivo in human cells. Moreover, by using recombinant mutated PCNA, we could demonstrate that pol lambda interacts with both the interdomain-connecting loop and the nearby hydrophobic pocket on the anterior of PCNA and that critical residues within a helix-hairpinhelix domain of pol lambda, important for proper DNA primer binding, are also involved in the enzymes interaction with PCNA. Finally, we showed that the tumor suppressor protein p21WAF1/CIP1 can efficiently compete in vitro with pol lambda for binding to PCNA. Given the high rate of frameshift mutations induced by pol lambda and its ability to bypass abasic sites, accurate regulation of pol lambda activity by PCNA and p21 concerted action might be important for preventing genetic instability.
- We enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). The tdt activity was strongly influenced by the nature of the 3-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3-overhang of about 9 12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3-ssDNA end by two alternative mechanisms. Most importantly we found that the PCNA was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally RP-A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that PCNA and RP-A can coordinate the polymerase and the terminal transferase activities of pol lambda.
- Improvement of Pab Pol B (already marketed by MPBiomedicals under the Isis brand name.
Experiments to optimize PabPol B incubation buffer, indicated that PCR performances and fidelity parameters were the highest in presence of 20mM Tris-HCl, pH = 9,0, 1.5mM MgSO4, 25mM KCl, 10mM (NH4)2 SO4 and 40µM of each dNTP. Under these conditions, the error rate was 0.66x10-6 mutation/nucleotide/duplication (m/n/d), similar to Pfu Pol B, issued from P. furiosus. Fidelity is 40 x higher than classical Taq DNA Pol whose error rate is 24x10-6 m/n/d. Fidelity of PabPol B depends on concentrations of MgSO4, as error rate increases to 1.39x10-6 m/n/d at 3mM MgSO4. Fidelity of PabPol B also depends on dNTPs concentration, as error rate increases from 0.66x10-6 m/n/d to 1.41x10-6m/n/d and 3.05x10-6m/n/d, when 100µM and 200µM of each dNTP are used respectively.
Variation of ionic strength has an obvious effect on fidelity of PabPol B. When KCl concentration decreases from 25mM to 10mM, the error rate increases from 0.66x10-6m/n/d to 1.25x10-6m/n/d. Moreover, if (NH4)2 SO4 concentration is brought to 0 mM, the error rate is 2.0 x 10-6 m/n/d. This value drastically increases to 18.7x10-6 when dNTPs are brought from 40 µM to 200 µM each. The effect of ionic strength was shown to behave similarly on PCR performances of PabPol B. The optimal composition of the PCR incubation buffer has been modified. The decrease of ionic strength, from 25mM to 10mM KCl or from 10mM to 0mM (NH4)2 SO4 , simply leads to inefficient amplification, in parallel with lower fidelity. That dependence of the accuracy and efficiency of elongation and amplification, on ionic strength, have conducted our subsequent studies on PabPol D.
The RF-C/PCNA complex enhanced the speed of PabPol B in vitro. That represents a big step for the development of a Proofreading Ultra Fast PCR complex. Shorter times of elongation still have to be explored.
We have shown that a blend of PabPol B 3exo(+)/ 3exo (-) at a ratio 50/1, amplifies up to 28. kb on lambda DNA, but with peculiar requirements of salt conditions (20 mM Tris-HCl pH = 9.0; 60 mM KCl). No evidence was detected on PCR assays made on a 21.7kb that the presence of either PCNA or/and RF-C, can improve long range PCR, except that the non-specific bands migrating around 3-4kb get weaker in presence of RF-C. In all cases, presence of PCNA generates inhibition even when associated with clamp loader RF-C. Further experiments still have to be made to show whether replication factors can stabilise PabPol B on the DNA strand for full length amplification up to 30-40kb.
- Pab Pol D as molecular biology tool.
PabPol D was shown to prefer primed DNA when performing single run on either circular or linear M13 DNA, but not exclusively, unlike PabPol B. Single run assays with PabPol D were successfully performed in conditions (20mM Bis Tris pH 6.8; 10mM MgCl2; 1mM DTT; 0.4 mg/ml BSA) which cannot be adapted to PCR assays, knowing that a minimal pH = 8.3 is required to avoid depurination at denaturation steps and that 1.5mM MgCl2 is required to reduce misincorporations. First PCR assays using PabPol D were performed on lambda DNA, using the optimised incubation buffer developed for PabPol B. PabPol D showed a bigger flexibility to variation of ionic strength than PabPol B, which was inefficient at 10mM KCl. On the contrary, at 10mM KCl and 10mM Tris HCl. PabPol D remained as efficient as in higher ionic strength. Nevertheless, absence of either KCl or (NH4)2 SO4, drastically reduced the efficiency of PabPol D, but this absence of KCl can be balanced by higher concentration of Tris HCl up to 25mM and (NH4)2 SO4. PCR assays in similar conditions were successfully performed on a human actin gene. In parallel, PabPol D did not stand higher temperatures than 95°C. It presented a much lower sensitivity than PabPol B and failed in Ultra Fast PCR assays, being unable to amplify a 400 bp under the required 45 seconds of elongation per cycle. At this state, no inhibition or activation has been observed when Pab PCNA was added to the PCR using PabPol D, while addition of RF-C still decreased the sensitivity of PabPol D. PabPol D displayed high efficiency in PCR on all types of template, showing less dependance to ionic strength than PabPol B which disliked low ionic strength.
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:
- Both Pabpols are DNA polymerizing enzymes exclusively;
- Their DNA binding properties as tested in gel shift competition assays indicated that PabpolD has a preference for a primed template;
- PabPolD is a primerdirected DNA polymerase independently of the primer composition whereas PabpolB behaves as an exclusively DNA primer-directed DNA polymerase;
- PabPCNA is required for PabpolD to perform efficient DNA synthesis but not PabpolB;
- PabpolD, but not PabpolB, contains strand displacement activity;
- In the presence of PabPCNA, however, both Pabpols D and B show strand displacement activity; and
- We show that the direct interaction between PabpolD and PabPCNA is DNAdependent.
Our data imply that PabPolD might play an important role in DNA replication likely together with PabpolB, suggesting that archaea require two DNA polymerases at the replication fork.
Dissemination: results already published in J. mol. Biol (2005)
Expected benefits: preliminary characterization of the polymerases prior to commercial development.
DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+ -dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we described the first genetic analysis of archaeal DNA ligase function. We showed that the H. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that H. volcanii also encodes an NAD+ -dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.
-We describe the identification of a chromosomal DNA replication origin (oriC) from the hyperthermophilic archaeon Sulfolobus solfataricus (subdomain of Crenarchaeota). A candidate oriC was mapped within a 1.12-kb region located between the two divergently transcribed MCM- and cdc6-like genes. We demonstrated that plasmids containing the Sulfolobus oriC sequence and a hygromycin-resistance selectable marker were maintained in an episomal state in transformed S. solfataricus cells under selective pressure. The proposed location of the origin was confirmed by 2-D gel electrophoresis experiments. This is the first report on the functional cloning of a chromosomal oriC from an archaeon and represents an important step toward the reconstitution of an archaeal in vitro DNA replication system.
- We report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 3 Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.
- We report that a Cdc6 (cell-division control)- like factor from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as SsoCdc6-2) has a modular organization of its biological functions. A reliable model of the SsoCdc6-2 three-dimensional structure was built up, based on the significant sequence identity with the Pyrobaculum aerophylum Cdc6 (PaeCdc6), whose crystallographic structure is known. This allowed us to design two truncated forms of SsoCdc6-2: the delta-C (residues 1 297, molecular mass 35 kDa) and the delta-N (residues 298 400, molecular mass 11 kDa) proteins. The delta-C protein contains the nucleotide-binding Rossmann fold and the Sensor-2 motif (Domains I and II in the PaeCdc6 structure), and retains the ability to bind and hydrolyse ATP. On the other hand, the delta-N protein contains the C-terminal WH (winged helix)-fold (Domain III), and is able to bind DNA molecules and to inhibit the DNA helicase activity of the SsoMCM (mini-chromosome maintenance) complex, although with lesser efficiency with respect to the full-sized SsoCdc6-2. These results provide direct biochemical evidence that the Cdc6 WH-domain is responsible for DNA-binding and inhibition of MCM DNA helicase activity. Moreover, it was demonstrated that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities.
- The biochemical characterization of Sso Cdc6-1 and Cdc6-3 has been done. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubblecontaining synthetic oligonucleotides. Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3.
- We report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity.
- Resveratrol, a natural compound found in many dietary plants and in red wine, plays an important role in the prevention of many humanpathological processes, including inflammation, atherosclerosis and carcinogenesis. Here, we present the first detailed biochemical investigation on the mechanism of action of resveratrol towards mammalian pols. Our results suggest that specific structural determinants of the resveratrol molecule are responsible for selective inhibition of different mammalian pols, such as the family B pol a and the family X pol.
Moreover, the resveratrol derivative trans-3,5-dimethoxy-4-hydroxystilbene, can inhibit pols and also suppress the in vitro SV40 DNA replication. The potency of inhibition is similar to that of aphidicolin, an inhibitor of the three replicative pols a, d and e. Our findings establish the necessary background for the synthesis of resveratrol derivatives having more selective and potent antiproliferative activity.
- More than 90% of leukemic cells in acute lymphocytic leukemia and approximately 30% of leukemic cells in the chronic myelogenous leukemia crisis show elevated TDT activity. This finding is connected to a poor prognosis and response to chemotherapy and reduced survival time. On the other hand, recent data indicated that TDT is not the only terminal deoxyribonucleotidyl transferase in mammalian cells. Its close relative, DNA polymerase l, can synthesize DNA both in a template-dependent (polymerase) and template-independent (terminal deoxyribonucleotidyl transferase) fashion. DNA polymerase l might be involved in the nonhomologous end-joining recombinational repair pathway of DNA double-strand breaks. Here, we report the characterization of the mechanism of action of three diketo hexenoic acid (DKHA) derivatives, which proved to be extremely selective for the terminal deoxyribonucleotidyl transferase activity of DNA polymerase l and TDT. They seem to be the first non nucleoside-specific inhibitors of mammalian terminal transferases reported. Moreover, the DKHA analog 6-(1-phenylmethyl- 1H-indol-3-yl)-2,4-dioxo-5-hexenoic acid (RDS2119) was not toxic toward HeLa cells (CC50 >100 mM), whereas it showed significant cytotoxicity against the TDT+ leukemia cell line MOLT-4 (CC50 = 14.9mM), thus having the potential to be further developed as a novel antitumor agent.
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