Volume 28, Issue 4, Pages 638-651 (November 2007) Sae2 Is an Endonuclease that Processes Hairpin DNA Cooperatively with the Mre11/Rad50/Xrs2 Complex  Bettina M. Lengsfeld, Alison J. Rattray, Venugopal Bhaskara, Rodolfo Ghirlando, Tanya T. Paull  Molecular Cell  Volume 28, Issue 4, Pages 638-651 (November 2007) DOI: 10.1016/j.molcel.2007.11.001 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Expression and DNA-Binding Activity of Recombinant Sae2 (A) Schematic representation of full-length wild-type Sae2 and mutants G270D, 5D (residues indicated changed to aspartate), 5A (residues indicated changed to alanine), ΔN (Δ aa 21–173), and ΔC (Δ aa 251–345). (B) SDS-PAGE of purified Sae2 wild-type and mutant proteins, ∼750 ng total protein each. (C) Wild-type (WT) and mutant Sae2 proteins were incubated with a 249 bp double-stranded DNA substrate and analyzed in a 8% native polyacrylamide gel in the presence of WT or R20M (RM) MRX complex. Protein-DNA complex 1 and complex 2 are indicated (described in text). Molecular Cell 2007 28, 638-651DOI: (10.1016/j.molcel.2007.11.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Nuclease Activities of MR and MRX Complexes (A) MRX was incubated with a 5′ 32P-labeled 46 bp DNA substrate containing a 4 nt recessed 3′ end in 1 mM MnCl2 or 5 mM MgCl2 with wild-type Sae2 protein as indicated. (B) The Rad50S MR(R20M)X complex was assayed in comparison to the wild-type complex as in (A) in 1 mM MnCl2. (C) The nuclease-deficient complexes M(D16A)R and M(Mre11-3)R were assayed in comparison to the wild-type MR complex as in (A) in 1 mM MnCl2. Molecular Cell 2007 28, 638-651DOI: (10.1016/j.molcel.2007.11.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Sae2 Cleaves Hairpin DNA (A) Wild-type MR and Sae2 were incubated with a 3′ 32P-labeled hairpin DNA substrate with 1 mM MnCl2 and 0.5 mM ATP as indicated and separated in a denaturing polyacrylamide gel. Substrate was also incubated with Mung Bean nuclease as a control to show the location of hairpin cut at the tip (MB). Numbers in the “M” lane indicate the positions of DNA standards run in the same gel. (B) Reactions were performed as in (A) with Sae2 only, 5 mM MgCl2, and internally labeled hairpin substrates as shown. (C) Reactions were performed with substrates identical to those in (B) except lacking the hairpin loops. Molecular Cell 2007 28, 638-651DOI: (10.1016/j.molcel.2007.11.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Sae2 and MRX Cooperatively Cleave Hairpin Structures (A) Wild-type MRX and Sae2 were incubated in 5 mM MgCl2 with an internally 32P-labeled hairpin DNA substrate as in Figure 3B. The primary cleavage products (arrow) are not cut at the tip of the hairpin but in the single-stranded overhang as shown in the diagram. MB control as in Figure 3. (B) Reactions were performed as in (A) but with substrates lacking the hairpin loop. (C) Wild-type MRX and Sae2 were incubated in 5 mM MgCl2 with an internally 32P-labeled hairpin DNA substrate with the opposite polarity compared to the substrate shown in (A). (D) Wild-type MRX and wild-type and mutant Sae2 proteins were incubated in 5 mM MgCl2 with an internally 32P-labeled hairpin DNA substrate as in (B). (E) Wild-type Sae2 and wild-type MRX and Rad50S MR(R20M)X complexes were assayed as in (A). Molecular Cell 2007 28, 638-651DOI: (10.1016/j.molcel.2007.11.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Mre11 Exonuclease Activity Facilitates Sae2 Hairpin Removal (A) An internally 32P-labeled double-hairpin DNA substrate was incubated first with wild-type MRX in 1 mM MnCl2 for 20 min; Sae2 was then added with 5 mM MgCl2, and the reactions were continued for an additional 30 min before separation on a denaturing polyacrylamide gel. MB control as in Figure 3. (B) Reactions were performed as in (A) but with wild-type MR and Mre11 nuclease-deficient complexes M(D16A)R and M(Mre11-3)R as indicated. (C) Hairpin cleavage assays as in (A) except that the substrate has a nick instead of a gap. (D) Model of MRX/Sae2 hairpin processing. Dotted arrow represents predicted MRX/Sae2-independent cleavage of the cruciform; circle-like object represents MRX 3′ to 5′ exonuclease activity; bold arrow represents Sae2 cleavage. The location of the cruciform cleavage site is assumed to be at the base of the cruciform as shown, but this has not been demonstrated in vivo. Molecular Cell 2007 28, 638-651DOI: (10.1016/j.molcel.2007.11.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Sae2 Cleaves Single-Stranded DNA in Branched DNA Structures (A) Diagram of DNA substrates 1–4 with locations of 32P labels (asterisks) and summary of predominant cleavage sites (arrows) as shown. (B) Wild-type and mutant Sae2 proteins were incubated with Substrate 1 in 5 mM MgCl2 as indicated. (C) Reactions with wild-type Sae2 on Substrates 1, 2, and 3 as in (B). (D) Wild-type Sae2 protein was incubated as in (B) with Substrate 4. (E) Wild-type Sae2 protein incubated as in (B) with substrates 1, 5, and 6. Molecular Cell 2007 28, 638-651DOI: (10.1016/j.molcel.2007.11.001) Copyright © 2007 Elsevier Inc. Terms and Conditions