Gap filling activities of Pseudomonas DNA ligase D (LigD) polymerase and functional interactions of LigD with the DNA end-binding Ku protein.
Many bacterial pathogens, including Pseudomonas aeruginosa, have a nonhomologous end joining (NHEJ) system of DNA double strand break (DSB) repair driven by Ku and DNA ligase D (LigD). LigD is a multifunctional enzyme composed of a ligase domain fused to an autonomous polymerase module (POL) that adds ribonucleotides or deoxyribonucleotides to DSB ends and primer-templates. LigD POL and the eukaryal NHEJ polymerase lambda are thought to bridge broken DNA ends via contacts with a duplex DNA segment downstream of the primer terminus, a scenario analogous to gap repair. Here, we characterized the gap repair activity of Pseudomonas LigD POL, which is more efficient than simple templated primer extension and relies on a 5'-phosphate group on the distal gap strand end to confer apparent processivity in filling gaps of 3 or 4 nucleotides. Mutations of the His-553, Arg-556, and Lys-566 side chains implicated in DNA 5'-phosphate binding eliminate the preferential filling of 5'-phosphate gaps. Mutating Phe-603, which is imputed to stack on the nucleobase of the template strand that includes the 1st bp of the downstream gap duplex segment, selectively affects incorporation of the final gap-closing nucleotide. We find that Pseudomonas Ku stimulates POL-catalyzed ribonucleotide addition to a plasmid DSB end and promotes plasmid end joining by full-length Pseudomonas LigD. A series of incremental truncations from the C terminus of the 293-amino acid Ku polypeptide identifies Ku-(1-229) as sufficient for homodimerization and LigD stimulation. The slightly longer Ku-(1-253) homodimer forms stable complexes at both ends of linear plasmid DNA that protect the DSBs from digestion by 5'- and 3'-exonucleases.
Zhu
Hui
H
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D020836:Protein Structure, Quaternary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase