{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Zhu H"],"funding":["NIGMS NIH HHS"],"pagination":["4815-25"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC2836087"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["285(7)"],"pubmed_abstract":["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."],"journal":["The Journal of biological chemistry"],"pubmed_title":["Gap filling activities of Pseudomonas DNA ligase D (LigD) polymerase and functional interactions of LigD with the DNA end-binding Ku protein."],"pmcid":["PMC2836087"],"funding_grant_id":["GM63611","R01 GM063611"],"pubmed_authors":["Shuman S","Zhu H"],"additional_accession":[]},"is_claimable":false,"name":"Gap filling activities of Pseudomonas DNA ligase D (LigD) polymerase and functional interactions of LigD with the DNA end-binding Ku protein.","description":"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.","dates":{"release":"2010-01-01T00:00:00Z","publication":"2010 Feb","modification":"2024-11-13T15:56:03.071Z","creation":"2019-03-27T00:29:13Z"},"accession":"S-EPMC2836087","cross_references":{"pubmed":["20018881"],"doi":["10.1074/jbc.M109.073874","10.1074/jbc.m109.073874"]}}