Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes

Abstract

Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.

Figure 1. Human, Yeast, and Archaeal M/R Complexes Exhibit Adenylate Kinase Activity

(A) Diagram of adenylate kinase reaction. (B) hsM/R complexes incubated with [γ-³²P]ATP ± AMP as indicated and the reactions separated by TLC. Positions of the [γ-³²P]ATP substrate and the [β-³²P]ADP and [³²P_i] products are shown. (C) pfM/R complexes assayed as in (B). (D) scM/R complexes assayed as in (B). (E) RecA (150 nM), BSA (150 nM), and hsRad54 (100 nM) incubated with [γ-³²P]ATP and AMP and characterized as in (B).

Figure 2. M/R Adenylate Kinase Activity Is Adenine Specific and Reversible

(A) Diagram of the adenylate kinase reaction with [α-³²P]AMP. (B) scM/R complexes incubated with [α-³²P]AMP and ATP and the reactions separated by TLC. Positions of the [α-³²P]AMP substrate and the [α-³²P]ADP and [α-³²P]ATP products are shown. (C) scM/R complexes assayed as in (B) except that various nucleotides were used in place of ATP as indicated. (D) scM/R complexes assayed with [γ-³²P]ATP and various nucleotides as indicated and characterized as in (B). (E) scM/R complexes assayed as in (D) with various metal cations (10 mM) as indicated; the amounts of [³²P_i] product are shown. (F) scM/R complexes assayed as in (E); the amounts of [β-³²P]ADP product are shown.

Figure 3. Adenylate Kinase Inhibitors Block the Adenylate Kinase Activity of M/R Complexes

(A) scM/R complexes incubated with [γ-³²P]ATP and AMP plus Ap₄A, Ap₅A, or Ap₆A as indicated and ATPase activity measured by the release of [³²P_i]. (B) scM/R complexes assayed as in (A) and adenylate kinase activity measured by the production of [β-³²P]ADP. (C) scM/R complexes assayed as in (A) plus Ap₃A or Ap₅A as indicated. (D) scM/R complexes assayed as in (B) plus Ap₃A or Ap₅A as indicated. (E) pfM/R and hsM/R assayed as in (A) with 5 μM Ap₅A as indicated. (F) pfM/R and hsM/R assayed as in (B) with 5 μM Ap₅A as indicated.

Figure 4. Mutation of the Rad50 Signature Motif Reduces Adenylate Kinase Activity by hsM/R and scM/R

(A) pfM/R wild-type and mutant (Rad50 S793R) complexes incubated with [γ-³²P]ATP and AMP and the reactions separated by TLC as in Figure 1B. (B) hsM/R wild-type and mutant (Rad50 S1202R) complexes assayed as in (A). (C) scM/R wild-type and mutant (Rad50 S1205R) complexes assayed as in (A). (D) Time course of ATPase activity by hsM/R complexes (300 nM) in the absence of AMP. (E) Time course of ATPase activity by hsM/R complexes as in (D) but in the presence of AMP. (F) Time course of adenylate kinase activity by hsM/R complexes in the presence of AMP. (G–I) The equivalent reactions as in (D)–(F), respectively, except performed with scM/R (2.5 μM).

Figure 5. Mutation of the Rad50 Signature Motif Inhibits Meiosis and Telomere Maintenance

(A) Diploid S. cerevisiae strains with combinations of Rad50 wild-type (WT), rad50 deletion (Δ), or rad50-S1205R (S1205R) alleles were induced to sporulate. The efficiency of sporulation was measured by random spore analysis; values shown are relative to the wild-type diploid. (B) Telomere length was measured in haploid strains expressing wild-type, rad50, or rad50-S1205R alleles (left panel). Genomic DNA from the wild-type strain was loaded undiluted (lane 1), diluted 1:5 (lane 2), or diluted 1:10 (lane 5). Genomic DNA from the rad50 and rad50-S1205R strains are loaded in lanes 3 and 4, respectively. Lanes 6–10 contain the same pattern of samples from a second group of isolates. The blot was probed with a telomere-specific probe. The same blot (right panel) is shown after stripping and reprobing with CEN4 to confirm equivalent gel loading.

Figure 6. Adenylate Kinase Activity Is Required for M/R-Mediated DNA Tethering

(A) Xenopus egg extracts incubated for 20 min with buffer, Ap₅A, or Ap₃A as indicated. Biotinylated DNA (0.6 × 10¹¹ ends/μl) and nonbiotinylated radioactive DNA (DNA*; 3 × 10¹¹ ends/μl) were added, and then biotinylated DNA was isolated and the associated radioactivity counted by scintillation. (B) ATM and Mre11 association with DNA was monitored by western blot in DNA-bound fractions using specific antibodies directed against ATM or Mre11. The equivalent of 100 ng of DNA was loaded on the gel. (C) scM/R complexes (30 nM) incubated with ³²P-labeled linear DNA (2.5 kb, 1 nM) in the presence of an unlabeled, biotinylated DNA fragment attached to streptavidin-coated magnetic beads. The percentage of labeled DNA associated with the beads is shown as an average of two experiments, with standard deviation as shown. (D) Reactions performed as in (C) except with scM/R(S1205R) and scM/R(K40A) mutant complexes as indicated.

Figure 7. Human and Yeast M/R Complexes Exhibit Reverse Adenylate Kinase Activity

(A) Diagram of adenylate kinase reaction starting with ATP hydrolysis (ATPase) and the reverse adenylate kinase reaction (AK). (B) Wild-type or scM/R(S1205R) complexes incubated with [α-³²P]ATP as indicated and analyzed by TLC. The positions of the [α-³²P]ATP substrate and [α-³²P]ADP and [α-³²P]AMP products are shown. (C) Wild-type or hsM/R(S1202R) complexes assayed as in (B). (D and E) scM/R complexes (2.5 μM) assayed with [α-³²P]ATP in the presence of varying amounts of linear DNA as indicated. The moles of [α-³²P]AMP produced, indicating adenylate kinase activity, are shown in (D) and the moles of [α-³²P]ADP produced, indicating ATP hydrolysis, are shown in (E).