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Search: authors:"Laurentino Villar"

7 papers found.
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Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in a number of linear genomes of viruses, linear plasmids and mobile elements. By this mechanism, a so-called terminal protein (TP) primes replication and becomes covalently linked to the genome ends. Bam35 belongs to a group of temperate tectiviruses infecting Gram-positive bacteria...

DNA stabilization at the Bacillus subtilis PolX core—a binding model to coordinate polymerase, AP-endonuclease and 3′-5′ exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been...

Dual Role of φ29 DNA Polymerase Lys529 in Stabilisation of the DNA Priming-Terminus and the Terminal Protein-Priming Residue at the Polymerisation Site

Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3′ terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA...

Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage ϕ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group...

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B...

Editing of misaligned 3′-termini by an intrinsic 3′–5′ exonuclease activity residing in the PHP domain of a family X DNA polymerase

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolXBs), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3′–5′ exonuclease activity specialized in resecting unannealed 3′-termini in a gapped DNA substrate. Biochemical analysis of a...

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...