Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity

This paper shows that the protein-primed DNA polymerases encoded by bacteriophages Nf and GA-1, unlike other DNA polymerases, do not require unwinding or processivity factors for efficient synthesis of full-length terminal protein (TP)-DNA. Analysis of their polymerization activity shows that both DNA polymerases base their replication efficiency on a high processivity and on the capacity to couple polymerization to strand displacement. Both enzymes are endowed with a proofreading activity that acts coordinately with the polymerization one to edit polymerization errors. Additionally, Nf double-stranded DNA binding protein (DBP) greatly stimulated the in vitro formation of the TP-dAMP initiation complex by decreasing the K(m) value for dATP of the Nf DNA polymerase by >20-fold. Whereas Nf DNA polymerase, as the φ29 enzyme, is able to use its homologous TP as well as DNA as primer, GA-1 DNA polymerase appears to have evolved to use its corresponding TP as the only primer of DNA synthesis. Such exceptional behaviour is discussed in the light of the recently solved structure of the DNA polymerase/TP complex of the related bacteriophage φ29

Insights into the determination of the templating nucleotide at the initiation of φ29 DNA replication

Background: The initiation of replication in φ29 mainly occurs opposite the 3' penultimate nucleotide of the template. Results: Mutations in the TP residue Phe-230 changed the specificity for the templating nucleotide. Conclusion: The results suggest a role for Phe-230 in determining the templating nucleotide. Significance: The results shed light on the positioning of the template nucleotide in organisms that initiate with proteinpriming mechanism.Spanish Ministry of Science and Innovation Grant CSD2007–00015 (to M. S.), and a Institutional grant from the Fundación Ramón Areces of the Centro de Biología MolecularPeer Reviewe

Current modalities of difference in the encounter between aging and adolescents: vicissitudes of the grandparenthood as a construction

El presente trabajo tiene como finalidad presentar los hallazgos del proyecto (PPID:2018-2019):Modalidades actuales del encuentro entre envejescentes y adolescentes: vicisitudes de la función de abuelidad en curso, en continuidad con la investigación previa: “Función de abuelidad y transmisión intergeneracional en las configuraciones familiares actuales”, Facultad de Psicología. UNLP. En anteriores presentaciones hicimos referencia a líneas teóricas y metodológicas, acompañadas con las primeras aproximaciones de resultados obtenidos de las entrevistas realizadas. En esta ocasión nos proponemos trasmitir resultados del análisis de los datos y las conclusiones alcanzadas. Los hallazgos obtenidos de la Investigación previa, revelan que la abuelidad, en tanto función, opera en ambos sentidos de manera recíproca inscribiendo los efectos del encuentro en abuelos y nietos. La presente investigación surge a partir de interrogantes formulados al finalizar la investigación anterior. Los entrevistados expresaban diferencias en el encuentro con los nietos niños en relación a los nietos adolescentes. Desprendidos de la niñez los jóvenes proponen una nueva modalidad de encuentro que exige a la función de abuelidad una reformulación. A su vez que resignifican desde el encuentro presente y valorizan experiencias. El de la niñez y de otro momento vital del abuelo; construcción de ese tiempo pasado compartido para proyectarse en el porvenir. De tal modo que la función de abuelidad logra su continuidad en un trabajo de reformulación del tiempo significando a posteriori nuevas imágenes del ser abuelo/a.Eje temático: Psicología del desarrollo. Enfoques y producciones en el devenir subjetivo.Facultad de Psicologí

Involvement of phage ϕ29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication

To initiate ϕ29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related ϕ29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical ϕ29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of ϕ29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed

Caracterización funcional de las DNA polimerasas de los bacteriófagos Nf y GA-1. Estudio del mecanismo de iniciación de la replicación con proteína terminal

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-06-2008Summary φ29-like bacteriophages have been classified into three groups based on serological properties, DNA sequences and protein maps, the main representatives of each group being φ29 (group I), Nf (group II) and GA-1 (group III). Bacteriophage φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that initiate DNA replication using a protein as primer. φ29 DNA replication requires the formation of an heterodimer between the φ29 DNA polymerase and a free molecule of the terminal protein (TP). During initiation this polymerase catalyses the addition of dAMP to the OH group of Ser232 in the TP. It has been demonstrated that the same DNA polymerase that accomplishes initiation catalyses the elongation processively and coupled to strand displacement without the need of either processivity factors or helicase-like proteins. In this Thesis we have characterised the replicative protein-primed DNA polymerases encoded by bacteriophages Nf and GA-1. We show that these DNA polymerases, like the φ29 enzyme, base their replication efficiency on high processivity and on the ability to couple polymerization to strand displacement. To achieve this, they do not require any unwinding or processivity factors. Moreover, both enzymes are endowed with a proofreading activity that acts coordinately with the polymerization one to edit polymerization errors. Interestingly, whereas Nf DNA polymerase, like the φ29 enzyme, is able to use both TP and DNA as primer, GA-1 DNA polymerase appears to have evolved to use its corresponding TP as the only primer of DNA synthesis. On the other hand, recent crystallographic studies of φ29 DNA polymerase/TP complex have provided structural insights into the specific recognition between DNA polymerase and TP. Biochemical analyses of a chimerical φ29 DNA polymerase containing the GA-1 TPR1 subdomain show that the interaction between the DNA polymerase TPR1 subdomain and the intermediate domain of the TP confers the specificity for the recognition between both proteins, and that the major contribution to the parental TP recognition is carried out by the DNA polymerase. In addition, we have shown that the DNA polymerase is the main protein involved in the recognition of the homologous DNA binding protein p6 (DBP). We have also studied the protein-primed DNA replication process in bateriophage Nf. The results presented here show that the initiation of Nf DNA replication is directed by the third 3’ nucleotide of its linear template, recovering the two terminal nucleotides by a stepwise sliding-back and/or a jumping back mechanism. Furthermore, by means of chimerical TP proteins, constructed by swapping the priming domain of φ29 and Nf TPs, we show that this domain is the main structural determinant that dictates the internal 3’ nucleotide used as template during initiation. Additionally, replacement of a specific α-helix of the Nf TP priming domain by the corresponding one of φ29 TP, endows Nf DNA polymerase with the ability to catalyse the DNA-independent initiation reaction

The Essential Role of the 3¿ Terminal Template Base in the First Steps of Protein-Primed DNA Replication

Bacteriophages w29 and Nf from Bacillus subtilis start replication of their linear genomes at both ends using a proteinprimed mechanism by means of which the DNA polymerase initiates replication by adding dAMP to the terminal protein, this insertion being directed by the second and third 39 terminal thymine of the template strand, respectively. In this work, we have obtained evidences about the role of the 39 terminal base during the initiation steps of w29 and Nf genome replication. The results indicate that the absence of the 39 terminal base modifies the initiation position carried out by w29 DNA polymerase in such a way that now the third position of the template, instead of the second one, guides the incorporation of the initiating nucleotide. In the case of Nf, although the lack of the 39 terminal base has no effect on the initiation position, its absence impairs further elongation of the TP-dAMP initiation product. The results show the essential role of the 39 terminal base in guaranteeing the correct positioning of replication origins at the polymerization active site to allow accurate initiation of replication and further elongation.Spanish Ministry of Science and Innovation (BFU 2008-00215, CSD 2007-00015); Autonomous Community of Madrid (grant P-MAT-0283-0505); Fundación Ramón ArecesPeer Reviewe

The essential role of the 3' terminal template base in the first steps of protein-primed DNA replication.

Bacteriophages ϕ29 and Nf from Bacillus subtilis start replication of their linear genomes at both ends using a protein-primed mechanism by means of which the DNA polymerase initiates replication by adding dAMP to the terminal protein, this insertion being directed by the second and third 3' terminal thymine of the template strand, respectively. In this work, we have obtained evidences about the role of the 3' terminal base during the initiation steps of ϕ29 and Nf genome replication. The results indicate that the absence of the 3' terminal base modifies the initiation position carried out by ϕ29 DNA polymerase in such a way that now the third position of the template, instead of the second one, guides the incorporation of the initiating nucleotide. In the case of Nf, although the lack of the 3' terminal base has no effect on the initiation position, its absence impairs further elongation of the TP-dAMP initiation product. The results show the essential role of the 3' terminal base in guaranteeing the correct positioning of replication origins at the polymerization active site to allow accurate initiation of replication and further elongation

The encounter with the difference in the link between adolescent grandparents and grandchildren. Grandparenthood as construction

El presente trabajo tiene como finalidad presentar los hallazgos del proyecto en curso (PPID: 2018-2019) “Modalidades actuales del encuentro entre envejescentes y adolescentes: vicisitudes de la función de abuelidad”, en continuidad con la investigación previa “Función de abuelidad y transmisión intergeneracional en las configuraciones familiares actuales”, de la Facultad de Psicología (UNLP). En anteriores presentaciones hicimos referencia a líneas teóricas y metodológicas, acompañadas con las primeras aproximaciones de resultados obtenidos de las entrevistas realizadas. En esta ocasión nos proponemos trasmitir resultados del análisis de los datos y las conclusiones alcanzadas. Los hallazgos obtenidos de la investigación previa, revelan que la abuelidad, en tanto función, opera en ambos sentidos de manera recíproca inscribiendo los efectos del encuentro entre abuelos y nietos. La presente investigación surge a partir de interrogantes formulados al finalizar la investigación previa. Los entrevistados expresaban diferencias en el encuentro con los nietos niños en relación a los nietos adolescentes. Desprendidos de la niñez los jóvenes proponen una nueva modalidad de encuentro que exige a la función de abuelidad una reformulación.Based on our theoretical foundations, we consider it essential for both the old and the adolescent, to be able to sign a narcissistic contract with the culture they belong to, since they need to project themselves in a hopeful manner in the future, in a place where their culture can lodge them. Under these circumstances, in which the future remains uncertain, we will have to inquire about the arrangements and reformulations the subjects are confronted to, to continue this task of transmitting and innovating. From the analysis of the data obtained we found a marked difference in the transmission from grandparents to grandchildren. From their role of grandparents they make special reference, as in the previous investigation, to their bond based on pleasure, conversation; the transmission of their own knowledge of each subject. A new form of presence that allows to emphasize the present, to leave a mark there, in the here and now present, and not only b bequeathing, but as a betting on a future. They recognize the role of grandparents as a construction of mutual reciprocity, based on the encounter with the different and nourished by multiple exchanges. In such a manner that requires a re-significance and a transformation of the bond between subjects in a work of metabolism of difference (with the other), teenage grandchildren are visualized and perceived as subjects with autonomy and freedom and time in their different dimensions (chronological, maturing, logical). In the bond's own psychic work, as "contact and renewed finding of newness between two subjects" (Berenstein, 2004), the gap of generational difference becomes a subject-subject challenge, from which older adults express their creative and unique strategies to accommodate, "lodge" the difference by offering themselves, as stated by Jacques Derrida (2000), to the alien, to the other. And to the extent that it is the other, it questions and raises questions on their truths built in other times.Facultad de Psicologí

Phage φ29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication

Bacteriophages φ29 and Nf from Bacillus subtilis start replication of their linear genome at both DNA ends by a protein-primed mechanism, by which the DNA polymerase, in a template-instructed reaction, adds 5′-dAMP to a molecule of terminal protein (TP) to form the initiation product TP-dAMP. Mutational analysis of the 3 terminal thymines of the Nf DNA end indicated that initiation of Nf DNA replication is directed by the third thymine on the template, the recovery of the 2 terminal nucleotides mainly occurring by a stepwise sliding-back mechanism. By using chimerical TPs, constructed by swapping the priming domain of the related φ29 and Nf proteins, we show that this domain is the main structural determinant that dictates the internal 3′ nucleotide used as template during initiation