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.
Highlights
Hang double strand break (DSB) are unfaithful, because the DSB ends are extended by polymerases or resected by nucleases prior to sealing by ligase (3, 4)
This study of Pseudomonas ligase D (LigD) polymerase domain (POL) underscores the primacy of the gap filling functions of dedicated nonhomologous end joining (NHEJ) polymerases and the influence of the gap 5Ј-phosphate end, a theme elaborated initially for human pol (24, 27) and subsequently for mycobacterial LigD POL (12, 25)
4-nucleotide gap that has a 5Ј-phosphate on the downstream duplex. (ii) The RNA polymerase activity of Pseudomonas aeruginosa LigD POL (PaePOL), which is much faster than dNMP addition during the first catalytic cycle (13), but is self-inhibited with each further cycle of rNMP addition to a standard primer-template (14), is enhanced by a 5Ј-phosphate gap that stimulates gap closure with a 3- or 4-nucleotide RNA patch. (iii) The salutary effects of the gap 5Ј-phosphate on primer utilization, processivity, and resulting gap closure are mediated by a trio of conserved residues (His-553, Arg-556, and Lys-566) that directly coordinate the gap 5Ј-phosphate in the homologous MtuPOL1⁄7DNA complex (Fig. 1)
Summary
Hang DSBs are unfaithful, because the DSB ends are extended by polymerases or resected by nucleases prior to sealing by ligase (3, 4). Strates than with dNTPs. These results fortify the suggestion primer terminus engaged productively in the active site, both that LigD POL might fill in short 5Ј-overhangs with ribonucle- structures revealed the following: (i) coordination of the distal otides when repairing DSBs. 5Ј-phosphate anion by amino acid side chains of the enzyme; Abasic sites are one of the most common forms of DNA (ii) compression or distortion of the template single strand damage, and they can exert profound effects when polymerases within the gap segment; and (iii) stacking of the first nucleobase encounter an abasic lesion in the template DNA strand.
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