title: Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D.
abstract: Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3'-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5'-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhu
authors.forename: Hitesh
authors.initials: H
authors.lastname: Bhattarai
authors.forename: Han-Guang
authors.initials: HG
authors.lastname: Yan
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
authors.forename: Michael S
authors.initials: MS
authors.lastname: Glickman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000502:physiology*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases; C493173:DNA ligase D, Mycobacterium tuberculosis; D000072481:DNA Ligase ATP