title: Novel 3'-ribonuclease and 3'-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D.
abstract: Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3'-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3'-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2'-OH group on the penultimate nucleoside of the primer strand. Replacing the 2'-OH by a 2'-F, 2'-NH2, 2'-OCH3, or 2'-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3'-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3'-PO4 terminus, and (ii) the 3'-PO4 is hydrolyzed to a 3'-OH. The 3'-ribonuclease and 3'-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3'-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3'-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3'-ribonuclease and 3'-phosphatase reactions.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhu
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000502:physiology*; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012260:Ribonucleases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D001224:Aspartic Acid; D006639:Histidine; D004247:DNA; D001120:Arginine; D012321:DNA-Directed RNA Polymerases; D012260:Ribonucleases; D010744:Phosphoric Monoester Hydrolases; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D000409:Alanine; D005990:Glycerol