Strande NT et al. (2014), Requirements for 5'dRP/AP lyase activity in Ku.

XB-ART-49697

Nucleic Acids Res 2014 Jan 01;4217:11136-43. doi: 10.1093/nar/gku796.

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Requirements for 5'dRP/AP lyase activity in Ku.

Strande NT , Carvajal-Garcia J , Hallett RA , Waters CA , Roberts SA , Strom C , Kuhlman B , Ramsden DA .

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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.

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Species referenced: Xenopus laevis
Genes referenced: acin1 apcs hnrnpu pias1 prkdc vwf

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	Figure 1. Identification of adducting lysines within human Ku70. A. Description of the 5′dRP/AP lyase reaction with abasic site, and use of NaBH4 to trap the covalent intermediate. B. Domain map of human Ku70 showing the location of lysines or groups of lysines that were mutated to alanine (A) or arginine (R). vWA, von Willebrand factor type A domain; SAP, SAF-A/B, Acinus and PIAS. C. Representative reactions were performed with 1 nM radiolabeled 5′dRP substrate with 5 nM purified recombinant Ku heterodimer at 37°C for 5 min, with the Ku70 subunit either WT or mutated as described in panel B. Reactions were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE). In lane 8 substrate was treated with alkali (OH) to validate abasic site generation. D. Average lyase velocities were determined by incubating 5 nM purified recombinant Ku heterodimer and 1 nM AP (filled bar) or 5′dRP (open bar) substrates at 37°C, and expressed as a percentage of the velocity observed with WT heterodimer. Error bars are the standard deviation of triplicate determinations. E. Purified heterodimers were subjected to sodium dodecyl sulphate (SDS)-PAGE (SDS-PAGE) analysis and detected directly by SYPRO orange (top panel). Products of Schiff-base trapping assays (bottom panel) were detected by phosphorimaging after incubation of the heterodimer with radiolabeled 5′dRP substrate as in panel C, except reactions were supplemented with 5 mM NaBH4 and incubated for 10 min. F. EMSA was performed by incubating 1nM Ku with 1 nM radiolabeled 30 bp substrate for 15 min.
	Figure 2. Identification of adducting lysines within human Ku80. A. Domain map of human Ku80 showing the location of lysines or groups of lysines that were mutated to alanine (A). B. Average velocities were determined from reactions at 37°C with 5 nM purified recombinant Ku heterodimer and 1 nM AP (filled bar) or 5′dRP (open bar) substrates, and expressed as a percentage of the velocity observed with WT heterodimer. Error bars are the standard deviation of triplicate determinations. C. Purified heterodimers were subjected to SDS-PAGE analysis and detected directly by SYPRO orange (top panel). Products of Schiff-base trapping assays (bottom panel) were detected by phosphorimaging after incubation of the heterodimer with radiolabeled 5′dRP substrate as in Figure 1, panel C, except reactions were supplemented with 5 mM NaBH4 and incubated for 10 min. *, unknown adducting species. D. EMSA was performed by incubating 1 nM Ku with 1 nM radiolabeled 30 bp substrate for 15 min.
	Figure 3. Effect of DNA-PKcs on 5′dRP/AP lyase activity. A. 1 nM radiolabeled AP substrate containing an abasic site was incubated at 37°C for 10 min with 2 nM Ku or the 2A/6A mutant Ku described in Figure 2. 2nM DNA-PKcs was included as noted. Mg&ATP, reactions were supplemented with 5mM MgCl2 and 100 μM ATP. Average velocities and standard deviations were determined from three independent experiments, and expressed relative to the activity observed with WT Ku heterodimer alone. B. Reactions were performed as in panel A, but supplemented with NaBH4, and analyzed by SDS-PAGE and phosphorimaging. *, unknown adducting species.
	Figure 4. AP lyase activity of Ku from model organisms. A. Alignment of Ku70 from different species. The start of the vWA domain is noted. K31 and flanking aromatics (targeted for mutation in Figure 5) are boxed. B. Reactions were performed with 1 nM radiolabeled AP substrate with 5 nM purified recombinant Ku heterodimer at 37°C for 2.5 min, using recombinant Ku heterodimer from S. cerevisiae (yWT), a yKu heterodimer with K29A and K161A substitutions in Ku70 (y2A), or a recombinant Ku heterodimer from X. laevis (fWT). In lane 6, substrate was digested with alkali (OH) to validate abasic site generation. Reactions were analyzed by denaturing PAGE. C. Average velocities were determined from reactions at 37°C with 5 nM purified recombinant Ku heterodimer and 1 nM AP substrate, and expressed as a percentage of the velocity observed with WT yeast Ku heterodimer (yWT). Error bars are the standard deviation of triplicate determinations. D. Reactions were performed as in panel B, but supplemented with NaBH4, and analyzed after a 10 min incubation by SDS-PAGE and phosphorimaging. E. EMSA was performed by incubating 1nM Ku with 1 nM radiolabeled 30 bp substrate for 15 min.
	Figure 5. Function of aromatic residues flanking K31. A and B. Representative reactions performed with 5 nM WT human Ku heterodimers or Ku heterodimers with Ku70 K160A, Ku70 K160A and Y30A, or Ku70 K160A, Y30A and Y32A substitutions. A. Reactions used the standard AP substrate and were incubated for 15 min. B. Reactions used a variant substrate with an AP site located an additional nucleotide further from the 5′ terminus, relative to the substrate used in panel A (see cartoon), and were incubated for 30 min. C. Average velocities and standard deviations were determined from three independent experiments, and expressed relative to the average velocity observed with a Ku heterodimer containing Ku70 with the K160A substitution.
	Figure 6. Model of the interaction between Ku70 K31 and an abasic site substrate. A and B. A cartoon representation of the human Ku heterodimer interacting with an abasic site substrate was modeled on the structure 1JEY (21). Five amino acids (pink) were appended to the most N-terminal residue resolved in Ku70 (G34), and the DNA altered to have an abasic site centered within a three nucleotide 5′ overhang. A. Ku70 is in green and Ku80 in cyan. The location of candidate nucleophiles identified in Figures 1 and 2 are shown in red. B. Ku70 K31 is shown in stick representation, with the dashed line showing the distance between nuclei predicted to participate in a Schiff base intermediate.

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Behmoaras, A tryptophan-containing peptide recognizes and cleaves DNA at apurinic sites. 1981, Pubmed