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Papers In Press, published online ahead of print May 15, 2005
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021
Corresponding Author: s-shuman{at}ski.mskcc.org
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-aa 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; (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 Asp50, Arg52 and His84, which we show are essential for the 3’ ribonuclease and 3’ phosphatase reactions.
J. Biol. Chem, 10.1074/jbc.M504002200
Submitted on April 13, 2005
Revised on May 11, 2005
Accepted on May 15, 2005
Novel 3' ribonuclease and 3' phosphatase activities of the bacterial NHEJ protein, DNA ligase D
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