Volume 8, Issue 5, Pages 1105-1115 (November 2001) Promotion of Dnl4-Catalyzed DNA End-Joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 Complexes  Ling Chen, Kelly Trujillo, William Ramos, Patrick Sung, Alan E. Tomkinson  Molecular Cell  Volume 8, Issue 5, Pages 1105-1115 (November 2001) DOI: 10.1016/S1097-2765(01)00388-4

Figure 1 Rad50/Mre11/Xrs2 Promotes Intermolecular DNA Joining by Dnl4/Lif1 (A) Left panel: After separation by SDS-PAGE, subunits of the purified Dnl4/Lif1 complex (1 μg) were stained with Coomassie blue. Right panel: the Dnl4/Lif1 complex (0.25 pmol) was incubated with [α-32P]ATP as described in Experimental Procedures. After separation by SDS-PAGE, the labeled Dnl4-adenylate (Dnl4*) was detected by autoradiography. (B) After separation by SDS-PAGE, subunits of the purified Rad50/Mre11/Xrs2 complex (4 μg) were stained with Coomassie blue. Xrs2 consists of a series of bands because of phosphorylation (Usui et al., 1998). The positions of the molecular mass standards (BioRad) are indicated on the left. (C) A 400-bp DNA substrate with 5′ cohesive ends (0.75 nM) was incubated with Dnl4/Lif1 (6 nM) or T4 DNA ligase (Roche, 0.01 U) in the presence or absence of Rad50/Mre11/Xrs2 (9 nM). In the absence of Rad50/Mre11/Xrs2, the rate of ligation is 0.0004 ligation events/enzyme complex/min. In the presence of Rad50/Mre11/Xrs2, the rate of ligation is 0.006 ligation events/enzyme complex/min. (D) Dnl4/Lif1 (6 nM) was incubated with: lanes 3–5, the indicated individual subunits, 18 nM each; lanes 6–8, the indicated pairwise combination of purified subunits, 18 nM each; lane 9, purified Rad50/Mre11/Xrs2 complex, 9 nM; lane 10, Rad50 (18 nM), Mre11 (18 nM), and Xrs2 (9 nM). In assays with the individually purified subunits, the proteins were incubated on ice for 10 min prior to the addition of the DNA substrate and incubation at 25°C. Lane C, DNA substrate only. The positions of the linear DNA substrate, circular monomer, and linear dimer and trimer are indicated on the left. The arrow indicates the positions of higher oligomers. Molecular Cell 2001 8, 1105-1115DOI: (10.1016/S1097-2765(01)00388-4)

Figure 2 Effect of Rad50/Mre11/Xrs2 Concentration on Inter- and Intramolecular Joining Catalyzed by Dnl4/Lif1 Labeled 400 bp DNA substrate with 5′ cohesive ends (0.75 nM) and Dnl4/Lif1 (6 nM) were incubated for 60 min either without, lane 1, or with Rad50/Mre11/Xrs2; lane 2, 0.375 nM; lane 3, 0.75 nM; lane 4, 1.5 nM; lane 5, 3 nM; lane 6, 6 nM; lane 7, 9 nM; lane 8, 12 nM; lane 9, 18 nM; and lane 10, 24 nM. Lane C, DNA substrate only. The DNA molecules are indicated as in the legend to Figure 1. The amount of ligated linear products (black bars) and circular products (white bars) at different Rad50/Mre11/Xrs2 concentrations was quantitated by phosphorimager analysis and presented in a graph below the gel. Molecular Cell 2001 8, 1105-1115DOI: (10.1016/S1097-2765(01)00388-4)

Figure 3 Rad50/Mre11/Xrs2 Greatly Enhances the Initial Rate of Joining by Dnl4/Lif1 (A) Labeled 400 bp DNA substrate with 5′ cohesive ends (0.75 nM) was incubated with either Dnl4/Lif1 (4 nM) or human DNA ligase IV/XRCC4 (4 nM) and Rad50/Mre11/Xrs2 (9 nM) as indicated. The DNA molecules are indicated as in the legend to Figure 1. (B) Graphic representation of DNA joining assays after quantitation by phosphorimager analysis. The results shown are from three independent experiments. DNA substrate with 5′ cohesive ends incubated with Dnl4/Lif1 (4 nM) and Rad50/Mre11/Xrs2 (9 nM) in the presence of 3 mM Mn2+ and 10 mM Mg2+ (filled triangles); DNA substrate with 5′ cohesive ends incubated with Dnl4/Lif1 (4 nM) and Rad50/Mre11/Xrs2 (9 nM) in the presence of 10 mM Mg2+ (open triangles); DNA substrate with 3′ cohesive ends incubated with Dnl4/Lif1 (4 nM) and Rad50/Mre11/Xrs2 (9 nM) in the presence of 3 mM Mn2+ and 10 mM Mg2+ (filled squares); DNA substrate with 3′ cohesive ends incubated with Dnl4/Lif1 (4 nM) and Rad50/Mre11/Xrs2 (9 nM) in the presence of 10 mM Mg2+ (open squares); DNA substrate with 3′ cohesive ends incubated with Dnl4/Lif1 (4 nM) in the presence of 10 mM Mg2+ (circles). In the absence of Rad50/Mre11/Xrs2, the rate of ligation is 0.0004 ligation events/enzyme complex/min. In the presence of Rad50/Mre11/Xrs2, the rate of ligation is 0.005 ligation events/enzyme complex/min with the 5′ substrate and 0.008 ligation events/enzyme complex/min with the 3′ substrate. Molecular Cell 2001 8, 1105-1115DOI: (10.1016/S1097-2765(01)00388-4)

Figure 4 Interaction between Rad50/Mre11/Xrs2 and Dnl4/Lif1 Complexes (A) [32P]-Adenylated Dnl4/Lif1 or Cdc9 DNA ligase was incubated with glutathione Sepharose beads containing GST (GST), GST-Mre11 (GST-M), or GST-Mre11/Rad50/Xrs2 (GST-MRX) as described in Experimental Procedures. (B) [32P]-Adenylated Dnl4/Lif1 was incubated with glutathione Sepharose beads containing GST (GST), GST-Mre11/Rad50 (GST-MR), or GST-Mre11/Xrs2 (GST-MX). (C) Combinations of 6×His-Lif1 (His-Lif1), Mre11, and Xrs2 were incubated with nickel NTA agarose beads to pull down the interacting proteins as described in Experimental Procedures. After centrifugation, the supernatants (S) were removed and the beads washed with buffer (W) before being treated with 200 mM imidazole to elute bound proteins (E). Proteins in equivalent volumes of the supernatant (S), wash (W), and eluted (E) fractions were run in an SDS-polyacrylamide gel and then detected by Coomassie blue staining and either autoradiography or immunoblotting as indicated. Bands corresponding to Rad50 (R), GST-Mre11 (GST-M), Xrs2 (X), Mre11 (M), BSA, 6×His-Lif1 (His-Lif1), and labeled Dnl4 (Dnl4*) and Cdc9 (Cdc9*) are indicated on the right. Molecular Cell 2001 8, 1105-1115DOI: (10.1016/S1097-2765(01)00388-4)

Figure 5 Interaction of the Rad50/Mre11/Xrs2 Complex with Linear Duplex DNA Molecules (A) The labeled 500 bp DNA substrate with 3′ cohesive ends (50 fmol DNA ends) was preincubated with: no Rad50/Mre11/Xrs2, filled circles; 162.5 fmol Rad50/Mre11/Xrs2, open circles; 325 fmol Rad50/Mre11/Xrs2, filled squares; and 640 fmol Rad50/Mre11/Xrs2, open squares, prior to digestion with λ exonuclease as described in Experimental Procedures. (B) Nucleoprotein complexes formed between a 400 bp DNA molecule with 5′ cohesive ends and the Rad50/Mre11/Xrs2 complex were visualized by AFM as described in Experimental Procedures. Upper left panel, DNA alone; upper right panel, DNA and Rad50/Mre11/Xrs2 complex. Some of the DNA-bound Rad50/Mre11/Xrs2 complexes are highlighted by the arrows (2 × 2 μm scan); lower left panel, a Rad50/Mre11/Xrs2 complex bound to one end of a unit length DNA molecule; lower right panel, oligomerization of 4 unit length DNA molecules by Rad50/Mre11/Xrs2 complexes (250 × 250 nm images, zoomed in from 1 × 1 μm scan). The average height of the three DNA-bound Rad50/Mre11/Xrs2 complexes is 2.1 ± 0.1 nm. Molecular Cell 2001 8, 1105-1115DOI: (10.1016/S1097-2765(01)00388-4)

Figure 6 Effect of Hdf1/Hdf2 on Rad50/Mre11/Xrs2-Mediated Stimulation of Intermolecular DNA Joining by Dnl4/Lif1 (A) After separation by SDS-PAGE, subunits of the purified Hdf1/Hdf2 complex (2.5 μg) were stained with Coomassie blue. (B) Labeled 400 bp DNA substrate with 5′ cohesive ends (0.75 nM) was incubated with the indicated DNA ligase. In the upper panel, Dnl4/Lif1 (6 nM) and Hdf1/Hdf2 (1.5 nM) were incubated as indicated with Rad50/Mre11/Xrs2; lane 2, 0.375 nM; lane 3, 0.75 nM; lane 4, 1.5 nM; lane 5, 3 nM; lane 6, 6 nM; lane 7, 9 nM; lane 8, 12 nM; lane 9, 18 nM; lane 10, 24 nM. Lane C, DNA substrate only; Lane H, reaction contained Dnl4/Lif1 and Hdf1/Hdf2. In the lower panel, T4 DNA ligase (0.005 U, Roche) and Hdf1/Hdf2 (1.5 nM) were incubated as indicated with Rad50/Mre11/Xrs2; lane 3, 3 nM; lane 4, 6 nM. (C) Ligation of labeled DNA substrate with 5′ cohesive ends (0.75 nM) by Dnl4/Lif1 (40 nM) was carried out in the presence of 100 mM KCl. The reactions contained Rad50/Mre11/Xrs2 (12 nM), Hdf1/Hdf2 (1.5 nM), and human Ku70/Ku80 (1.5 nM) where indicated. The incubation time was 60 min for all reactions. The results from three independent assays were quantitated by phosphorimager analysis and presented in the graph below the gel panel. Molecular Cell 2001 8, 1105-1115DOI: (10.1016/S1097-2765(01)00388-4)