title: Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins.
abstract: Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+). Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3'-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.
authors.affiliation: Centro Nacional de Biotecnología-CSIC, Darwin 3, 28049 Madrid, Spain.
authors.forename: Paula P
authors.initials: PP
authors.lastname: Cardenas
authors.forename: Thomas
authors.initials: T
authors.lastname: Carzaniga
authors.forename: Sandro
authors.initials: S
authors.lastname: Zangrossi
authors.forename: Federica
authors.initials: F
authors.lastname: Briani
authors.forename: Esther
authors.initials: E
authors.lastname: Garcia-Tirado
authors.forename: Gianni
authors.initials: G
authors.lastname: Dehò
authors.forename: Juan C
authors.initials: JC
authors.lastname: Alonso
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D008345:Manganese; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; D011117:Polyribonucleotide Nucleotidyltransferase; D005090:Exodeoxyribonucleases; D004262:DNA Restriction Enzymes; C047879:RecN protein, Bacteria; C026600:2'-deoxyadenosine triphosphate