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Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing.

ABSTRACT: High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.

SUBMITTER: Wang H

PROVIDER: S-EPMC4215242 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:5'- and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH and 3'-PO4 or 2',3'-cyclic-PO4 ends are converted to 5'-PO4 and 3'-OH termini suitable for sealing by polynucleotide ligases. Here, we characterize Deinococcus radiodurans HD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5'-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3'-phosphatase and 2',3'-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis of p-nitrophenylphosphate. HD-Pnk is encoded by the LIG-PARG-HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential for Deinococcus growth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ?HD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitize Deinococcus to killing by mitomycin C.IMPORTANCE End healing is a process whereby nucleic acid breaks with "dirty" 3'-PO4 or 2',3'-cyclic-PO4 and 5'-OH ends are converted to 3'-OH and 5'-PO4 termini that are amenable to downstream repair reactions. Deinococcus radiodurans is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights Deinococcus HD-Pnk as a bifunctional 3'- and 5'-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

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Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing.

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