ABSTRACT
Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.
Subject(s)
DNA Damage , DNA Topoisomerases, Type II/metabolism , Phosphoric Diester Hydrolases/chemistry , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/chemistry , Animals , Catalytic Domain , DNA/chemistry , DNA/metabolism , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA End-Joining Repair , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins , Humans , Magnesium/chemistry , Mice , Mice, Knockout , Models, Molecular , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Phosphotyrosine/metabolism , Polymorphism, Single Nucleotide , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolismABSTRACT
Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) µ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol µ using the unpaired base adjacent to the downstream 5' phosphate even when there are available template sites further upstream of this position; this makes Pol µ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol µ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.
Subject(s)
DNA End-Joining Repair , DNA Polymerase beta/chemistry , DNA-Directed DNA Polymerase/chemistry , Animals , Cell Proliferation , DNA/chemistry , DNA Damage , Dose-Response Relationship, Radiation , Fibroblasts/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nucleotides/chemistry , Radiation, IonizingABSTRACT
The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5' deoxyribosephosphate (5'dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5'dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role.
Subject(s)
Antigens, Nuclear/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-Binding Proteins/chemistry , Animals , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Ku Autoantigen , Lysine/chemistry , Models, Molecular , Mutation , Ribosemonophosphates/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Xenopus Proteins/metabolism , Xenopus laevisABSTRACT
Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85 to 6%. End-processing by nucleases and polymerases is increased to compensate, although paths with the fewest number of steps to generate a substrate suitable for ligation are favoured. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors.
Subject(s)
DNA End-Joining Repair , HCT116 Cells , HumansABSTRACT
Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy.
