vector 48324  (Addgene inc)

Journal: Nucleic acids research

Article Title: The conserved XPF:ERCC1-like Zip2:Spo16 complex controls meiotic crossover formation through structure-specific DNA binding.

doi: 10.1093/nar/gky1273

Figure Lengend Snippet: Figure 1. Zip2, Zip4, and Spo16 form a complex. (A) Domain structure of Zip2, Zip4, and Spo16. Gray arrows indicate interactions identified by yeast two-hybrid and yeast three-hybrid analysis. The N-terminal do- main of Zip2 has been proposed to contain a WD40 -propeller domain (36), but the region is poorly conserved (Supplementary Figure S2) and modern structure-prediction algorithms do not support this assignment. HhH: helix-hairpin-helix, HhH2: tandem helix-hairpin-helix. (B) Yeast two-hybrid analysis of interactions between Zip2, Zip4, and Spo16. See Supplementary Figure S1A for complete results. (C) Yeast three-hybrid analysis. See Supplementary Figure S1B for complete results. (D) Size ex- clusion chromatography/multi-angle light scattering (SEC-MALS) anal- ysis of purified Zip2499–704:Spo16. The measured molecular weight (51.8 kDa) is consistent with a 1:1 heterodimer (molecular weight 49.8 kDa). (E) SDS-PAGE analysis of purified Zip2499–704:Spo16.

Article Snippet: To express TEV protease-cleavable, His6-tagged Spo16, full-length Spo16 was cloned into Addgene vector 48324 (contains Spectinomycin resistance and CloDF13 ori) using ligation-independent cloning.

Techniques: Chromatography, Multi-Angle Light Scattering, Purification, Molecular Weight, SDS Page

Journal: Nucleic acids research

Article Title: The conserved XPF:ERCC1-like Zip2:Spo16 complex controls meiotic crossover formation through structure-specific DNA binding.

doi: 10.1093/nar/gky1273

Figure Lengend Snippet: Figure 2. Structure of the Zip2499–704:Spo16 complex. (A) Domain schematic of Zip2 (truncated construct for crystallization) and Spo16. (B) Two views of the Zip2499–704:Spo16 dimer, with domains colored as in (A). In one view, secondary structure elements of both proteins’ central domains are labeled as in panel (C). See Supplementary Figure S3F for an overlay of the five crystallographically-independent views of the dimer. (C) Secondary structure of the Zip2 and Spo16 central domains (top) and HhH domains (bottom). (D) Overlay of the Zip2 HhH2 and Spo16 HhH domains. C r.m.s.d. = 2.65 ˚A over 34 atom pairs. HhH #1 comprises helices and separated by hairpin 1 (h1), and HhH #2 (not shared by Spo16) comprises helices and ε, separated by hairpin 2(h2). (E) Overlay of the Zip2 HhH2 domain (pink) with the HhH2 domain of H. sapiens XPF (PDB ID 1Z00; gray). C r.m.s.d. = 1.97 ˚A over 51 atom pairs. (F) Overlay of the Zip2 central domain (blue) with the Aeropyrum pernix XPF nuclease domain (gray) (PDB ID 2BGW) (79). C r.m.s.d. = 2.50 ˚A over 107 atom pairs. Left: close-up of the active site of A. pernix XPF (gray, with bound Mg2+ ion shown as a sphere) with the equivalent region of the Zip2 central domain (blue). Zip2′s DNA binding patch 1 (see Figure 4B) comprises residues R554, R559 and K568 (labeled in black).

Article Snippet: To express TEV protease-cleavable, His6-tagged Spo16, full-length Spo16 was cloned into Addgene vector 48324 (contains Spectinomycin resistance and CloDF13 ori) using ligation-independent cloning.

Techniques: Construct, Crystallization Assay, Labeling, Binding Assay

Journal: Nucleic acids research

Article Title: The conserved XPF:ERCC1-like Zip2:Spo16 complex controls meiotic crossover formation through structure-specific DNA binding.

doi: 10.1093/nar/gky1273

Figure Lengend Snippet: Figure 3. DNA binding by Zip2499–704:Spo16. Representative gel-shift (upper) and binding curve from triplicate experiments (lower) for Zip2499–704:Spo16 binding dsDNA (A), 5′-overhang (B), 3′-overhang (C), nicked (D), Holliday Junction (E), and nicked Holliday Junction (F) DNA substrates. Protein concentrations in each lane (left to right: 0, 0.625, 2.5, 5, 10, 20, and 40 M) are the same for all gels. Red asterisks indicate location of multiple shifted bands for the HJ substrate. Gray boxes indicate Kd val- ues for dsDNA, nicked DNA, and Holliday Junction DNA as measured by isothermal titration calorimetry (Supplementary Figure S4).

Article Snippet: To express TEV protease-cleavable, His6-tagged Spo16, full-length Spo16 was cloned into Addgene vector 48324 (contains Spectinomycin resistance and CloDF13 ori) using ligation-independent cloning.

Techniques: Binding Assay, Gel Shift, Isothermal Titration Calorimetry

Journal: Nucleic acids research

Article Title: The conserved XPF:ERCC1-like Zip2:Spo16 complex controls meiotic crossover formation through structure-specific DNA binding.

doi: 10.1093/nar/gky1273

Figure Lengend Snippet: Figure 4. Identification of DNA-binding surfaces of Zip2499–704:Spo16. (A) Model of the Zip2499–704:Spo16 complex bound to a nicked/overhang DNA, based on a structure of human Mus81:Eme1 in complex with a 5′-flap DNA (PDB ID 4P0R (83); see Supplementary Figure S6). The central domains are oriented equivalently to Figure 2A. In the model, the HhH domains of Zip2 and Spo16 have undergone a significant conformational change from their original orientation (semi-transparent ribbons) upon DNA binding (gray arrow). Gray circles indicate the locations of putative DNA-binding patches 1–4. See Supplementary Figure S7 for surface charge distribution. (B) Close-up view of patches 1 and 2 on the central domains of Zip2 and Spo16. See Supplementary Figure S7B for surface charge distribution. (C) Close-up view of patches 3 and 4 on the Zip2 HhH2 domain. See Supplementary Figure S7C for surface charge distribution. (D) Representative gel-shift assays for patch 1 mutants binding nicked DNA. See Supplementary Figure S9 for binding of 5′ overhang DNA and HJ DNA by these mutants. (E) Representative gel-shift assays for patch 2 mutants binding nicked DNA. (F) Representative gel-shift assays for patch 3 mutants binding nicked DNA.

Article Snippet: To express TEV protease-cleavable, His6-tagged Spo16, full-length Spo16 was cloned into Addgene vector 48324 (contains Spectinomycin resistance and CloDF13 ori) using ligation-independent cloning.

Techniques: Binding Assay, Gel Shift

Journal: Nucleic acids research

Article Title: The conserved XPF:ERCC1-like Zip2:Spo16 complex controls meiotic crossover formation through structure-specific DNA binding.

doi: 10.1093/nar/gky1273

Figure Lengend Snippet: Figure 5. Model for the role of Zip2:Spo16:Zip4 in meiotic crossover formation. Model for the function of Zip2:Zip4:Spo16 in meiotic re- combination. Initial strand invasion is mediated by Rad51 and Dmc1, and is continually counteracted by the dissolution activities of Sgs1, Top3 and Rmi1, resulting in SDSA. Recognition of specific DNA struc- tures by Msh4:Msh5 (pro-HJ; pink shading) and Zip2: stabilize the strand- invasion/D-loop intermediate. Inset: Potential role of Zip2:Spo16 in rec- ognizing unligated/nicked Holliday Junctions and directing the cleavage activity of Mlh1:Mlh3 to promote crossover formation. Additional protein complexes scaffolded by Zip4 may license assembly of the synaptonemal complex in coordination with crossover formation.

Article Snippet: To express TEV protease-cleavable, His6-tagged Spo16, full-length Spo16 was cloned into Addgene vector 48324 (contains Spectinomycin resistance and CloDF13 ori) using ligation-independent cloning.

Techniques: Dissolution, Activity Assay