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Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

In all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3′-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAProcomplex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNAProcomplex. In contrast with other retroviruses, we found that the interaction between tRNAProand the M-MuLV RNA template is restricted to the standard PBS interaction. In this binary complex, the viral RNA is highly constrained and the rest of tRNAProis rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAProlacking the post-transcriptional modifications is substituted for the natural tRNAProprimer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNAProprevent additional contacts between tRNAProand the U5 region of M-MuLV RNA.

Retroviruses contain a dimeric RNA consisting of two identical molecules of genomic RNA. The interaction between the two monomers is thought to occur near their 5'ends. We previously identified a region upstream from the splice donor site, comprising an autocomplementary sequence, responsible for the formation of dimeric HIV-1Lai RNA [Muriaux, D., Girard, P.-M., Bonnet-Mathonire, B., & Paoletti, J.(1995) J. Biol. Chem. 270,8209-8216]. This region appeared to be confined within a putative stem-loop structure. Here we report an in vitro model under conditions of low inioc strength. Two dimers of RNA 77-402 were identified as a function of temperature, and a significant difference was found in their thermostability. Dimer D55, formed at 55 degrees Celsius, is more stable than dimer D37, formed at 37 degrees C. RNase probing experiments confirm the involvement of a stem-loop structure in the dimerization process. In the monomer, the free G257-CGCGC262 sequence forms a loop in the 240-280 region of RNA 77-402, whereas this sequence is engaged in base pairing when D55 and D37 dimers are formed. Our results show that the loop-loop interaction of the autocomplementary G257CGCGC262 sequence, though hydrogen bonding, is responsible for the formation of dimer D37 and strongly suggest that D37 is a "kissing" complex. In contrast, in dimer D55, all the nucleotides of the two hairpin stems, 243-254/264-277, are involved in a complete interstrand interaction.

The DNA binding properties and effects on topoisomerase II of MePyGA, an anilinoacridine derivative bearing an N-methylpyrrolecarboxamide unit at position 1', have been compared with those of its precursor glycylanilinoacridine and the structurally related antileukaemic drug amsacrine. Electric linear dichroism spectroscopy reveals that MePyGA intercalates its acridine chromophore between DNA base pairs with a preference for GC-rich sequences, whereas both its structural analogue lacking the N-methylpyrrole unit and amsacrine intercalate into DNA without any strong sequence preference. The effects of the test drug on the catalytic activities of topoisomerase II were studied in vitro using purified calf thymus enzyme and 32P-labeled DNA. MePyGA stabilizes the topoisomerase II-DNA covalent complex and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. The removal of the N-methylpyrrole unit abolishes both the GC-preferential binding to DNA and the topoisomerase II-mediated DNA cleavage. MePyGA and amsacrine stimulate the cleavage of DNA by topoisomerase II at different places: cleavage stimulated by amsacrine is consistent with the expected adenine requirement at position +1 whereas the predominant sites of DNA cleavage stimulated by MePyGA contain a cytosine at position +/- 1. This is the first instance where an anilinoacridine derivative differing only by the nature of the substituent at position 1' has been found to affect the catalytic activity of topoisomerase II differently. The spectroscopic and biochemical data lead to the conclusion that two functional domains can be identified in MePyGA: its anilino group can be regarded as a skeletal core to which are connected (i) the tricyclic acridine moiety which represents the DNA-binding domain and (ii) the N-methylpyrrole moiety which constitutes the topoisomerase II-targeted domain. The structure of the substituent at position 1' of the anilinoacridine chromophore evidently determines the location of the sites of DNA cleavage by topoisomerase II. These findings provide guidance for the synthesis and development of new topoisomerase II-targeted antitumor anilinoacridine derivatives.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.

gamma-Carbolines are tricyclic aromatic compounds which intercalate into DNA base pairs and exhibit significant cytotoxic and antitumor activities. These compounds which are structurally related to ellipticine by deletion of an aromatic ring, induce DNA breaks in cultured L1210 cells. Since the mechanism of cytotoxic activity of ellipticines involves DNA topoisomerase II, this enzyme might also be a target for gamma-Carbolines. We have tested this hypothesis using an in vitro system containing purified enzyme and pBR322 DNA. The ability of nine derivatives to stabilize the DNA-enzyme covalent complex was studied and compared to their cytotoxicity. The four less cytotoxic compounds do not induce cleavable complex to a significant extent. In contrast, the two most cytotoxic gamma-Carbolines are the most efficient stabilizers of the cleavable complex. The last three compounds exhibit an intermediate cytotoxicity and cleavage activity. In the presence of gamma-Carbolines, cleavage occurs predominantly at a single site in pBR322 which is one of the cleavage sites observed with ellipticines. The cleavage position was determined at the nucleotide level. The increased DNA cleavage specificity observed with gamma-Carbolines suggests that a tricyclic system is as efficient as ellipticines for DNA topoisomerase II cleavage at DNA sequences involved specifically in cytotoxic response. The data presented support the hypothesis that DNA topoisomerase II is a target involved in the mechanisms of action of antitumor gamma-Carbolines.

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20–50 μM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo . Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

The antitumor drug VP-16 stabilizes the topoisomerase II-DNA covalent complexes formed in an intermediate step of the isomerization reaction. The location of the sites of formation of these complexes and their relative strength were studied in vitro using pBR322. Sequences alignment of the regions containing the 24 detectable sites allows to identify GCGCGC-(N) alpha-TGAC with 9 less than or equal to alpha less than or equal to 25 as the DNA sequence recognized by topoisomerase II to form a cleavable complex. Changes in the last two nucleotides of the sequence determine weaker complexes.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first trand transfer that requires base pairing of the R region at the 3’- end of the genomic RNA with the complementary r region at the 3’-end of minus strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the fulllength HIV-1 ssDNA in the absence or presence of NC.Our probing data obtained in the absence of NC, suggest weak contacts between the u5 region of ssDNA and the U3 region the genomic RNA. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Using the gel retardation assay, we analyzed the interaction of NC with each site. Taken together, our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the threedimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the annealing of ssDNA to the 3’- end of the genomic RNA requires NC activity and does not rely on a single pathway (zipper intermediate or kissing complex)., Les premier et second transferts de brin sont deux étapes essentielles de la transcription inverse du génome du virus de l’immunodéficience humaine de type 1 (VIH-1). De nombreuses études in vitro suggèrent que les transferts nécessitent l’action de la protéine de nucléocapside du VIH-1 (NC). Le premier transfert, se produisant de l’extrémité 5’ vers l’extrémité 3’ de l’ARN génomique du VIH-1, repose en grande partie sur un appariement ADN-ARN impliquant la région r de l’ADN strong-stop (ADNss) et la région R située à l’extrémité 3’ de l’ARN viral (ARN 3’UTR). Les structures, interactions et dynamiques qui gouvernent cet appariement sont mal connues. Jusqu’à notre étude, la formation de l’hybride ADN-ARN n’avait été étudiée in vitro qu’avec des courts acides nucléiques qui ne reflètent que partiellement les structures formées par l’ADNss et l’ARN 3’UTR dans le virus en cours de réplication. L’objectif principal de ma thèse a été de caractériser in vitro les mécanismes moléculaires qui gouvernent l’hybridation de l’ADNss avec l’ARN 3’UTR. Pour atteindre cet objectif, j’ai utilisé des méthodes de la biologie moléculaire et j’ai analysé la structure secondaire de l’ADNss entier avec trois sondes de structure (DNase I, mung bean nuclease et permanganate de potassium) : i) en l’absence de NC ; ii) en présence de NC ; iii) en présence de l’ARN 3’UTR.Les résultats obtenus nous ont permis d’être les premiers à déterminer in vitro la structure secondaire de l’ADNss entier du VIH-1 et à identifier dans celui-ci quatre sites sur lesquels se fixe préférentiellement la NC. A notre connaissance, les structures secondaires des ADNss d’autres rétrovirus n’ont pas été déterminées. Nos données structurales sont en faveur d’une structure secondaire de l’ADNss du VIH-1 constituée de trois tiges-boucles (u5, cpoly(A) et cTAR) et trois régions simple-brin en l’absence ou présence de NC. Notre analyse phylogénétique suggère que la structure secondaire de l’ADNss et les sites forts NC sont conservés parmi les différents groupes du VIH-1. Nos résultats suggèrent aussi qu’une partie de la région u5 de l’ADNss établit des interactions très faibles et probablement transitoires avec une partie de la région U3 de l’ARN 3’UTR en l’absence de NC. En réalisant des cinétiques d’hybridation et en utilisant deux ADNss mutés, nous avons montré que l’hybridation ADNss-ARN 3’UTR nécessite l’activité de la NC et que ce processus ne repose pas sur une seule voie d’initiation. Nos résultats supportent un modèle dans lequel la première étape est la fixation de la NC au niveau des quatre sites, ce qui va déclencher l’ouverture de la structure tridimensionnelle de l’ADNss et favoriser ainsi l’accessibilité de la région r ; la seconde étape étant la déstabilisation par la NC des structures secondaires ; la troisième étape étant l’appariement par la NC des régions complémentaires r et R. L’ensemble des résultats obtenus permet à mon équipe d’accueil d’initier de nouvelles études in vitro et ex vivo.

The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3’ R sequence of viral RNA (3’ UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3’ UTR; 4) the annealing of ssDNA to 3’ UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination.; Le 1er transfert de brin, étape cruciale de la transcription inverse impliquant la protéine de nucléocapside du VIH-1 (NC), repose sur l’appariement de la séquence r de l’ADN « strong stop » (ADNss) avec la séquence 3’ R de l’ARN viral (3’UTR) qui forme les tiges-boucles TAR et polyA. La séquence r est supposé former les tiges-boucles cTAR et cpolyA. Le transfert repose donc probablement sur l’hybridation de molécules structurées. La structure secondaire de l’ADNss n’a jamais été déterminée. L’objectif a été d’identifier les interactions et structures gouvernant l’hybridation de l’ADNss avec l’ARN 3’UTR. Les outils de la biologie moléculaire et trois sondes de structure ciblant l’ADN ont été utilisés pour atteindre cet objectif. Nos résultats sont les suivants : 1) l’ADN cTAR nu se replie sous la forme de deux conformations différentes qui sont en équilibre ; 2) la NC peut déplacer l’équilibre vers l’une des conformations et se fixer préférentiellement sur la boucle interne du cTAR ; 3) la NC est exigée pour former un hétéroduplex constitué de l’intégralité de l’ADNss et du 3’ UTR ; 4) l’hybridation ADNss-3’UTR peut être initiée à partir de plusieurs sites dans 0,2 mM MgCl2 ; 5) l’ADNss forme deux conformations en équilibre dans 0,2 mM MgCl2 et principalement une seule dans 2 mM MgCl2 ; 6) dans l’ADNss, la NC se fixe préférentiellement au niveau de la région simple-brin qui relie les tiges-boucles cTAR et cpolyA. Cette fixation joue probablement un rôle important dans l’hybridation des tiges-boucles ARN et ADN complémentaires. Notre étude permet de mieux comprendre la transcription inverse et la recombinaison qui dépend du transfert de brin interne.

The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3’ R sequence of viral RNA (3’ UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3’ UTR; 4) the annealing of ssDNA to 3’ UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination., Le 1er transfert de brin, étape cruciale de la transcription inverse impliquant la protéine de nucléocapside du VIH-1 (NC), repose sur l’appariement de la séquence r de l’ADN « strong stop » (ADNss) avec la séquence 3’ R de l’ARN viral (3’UTR) qui forme les tiges-boucles TAR et polyA. La séquence r est supposé former les tiges-boucles cTAR et cpolyA. Le transfert repose donc probablement sur l’hybridation de molécules structurées. La structure secondaire de l’ADNss n’a jamais été déterminée. L’objectif a été d’identifier les interactions et structures gouvernant l’hybridation de l’ADNss avec l’ARN 3’UTR. Les outils de la biologie moléculaire et trois sondes de structure ciblant l’ADN ont été utilisés pour atteindre cet objectif. Nos résultats sont les suivants : 1) l’ADN cTAR nu se replie sous la forme de deux conformations différentes qui sont en équilibre ; 2) la NC peut déplacer l’équilibre vers l’une des conformations et se fixer préférentiellement sur la boucle interne du cTAR ; 3) la NC est exigée pour former un hétéroduplex constitué de l’intégralité de l’ADNss et du 3’ UTR ; 4) l’hybridation ADNss-3’UTR peut être initiée à partir de plusieurs sites dans 0,2 mM MgCl2 ; 5) l’ADNss forme deux conformations en équilibre dans 0,2 mM MgCl2 et principalement une seule dans 2 mM MgCl2 ; 6) dans l’ADNss, la NC se fixe préférentiellement au niveau de la région simple-brin qui relie les tiges-boucles cTAR et cpolyA. Cette fixation joue probablement un rôle important dans l’hybridation des tiges-boucles ARN et ADN complémentaires. Notre étude permet de mieux comprendre la transcription inverse et la recombinaison qui dépend du transfert de brin interne.