Coomassie blue

Wild type arnA was PCR amplified from E. coli genomic DNA with NdeI and XhoI restriction site overhangs on the 5’ and 3’ ends, respectively, using primers 1F and 1R (See all primer details in Table S1), and cloned into the bacterial expression vector pColaDuet (EMD Millipore). Two serine point mutations were introduced at site 1 (H359S and H361S) using primers 2F and 2R. Two additional serine point mutations were introduced at site 2 (H592S and H593S) using primers 3F and 3R to generate the final arnA mutant containing a total of four histidine to serine mutations.
The arnA knockout strain was generated with the E. coli recombineering technique^{10 (link)}, using the pKD4 plasmid as a template for the selectable marker and BL21(DE3) as the parental strain. The forward and reverse primers, 4F and 4R, were designed to maintain the reading frame of arnB, which shares its start codon with the stop codon of arnA within the arn operon^{11 (link)} (also called pmrHFIJKLM operon^{12 (link)}). A slightly modified scheme was used to introduce the arnA mutant back into the arnA knockout strain at the original locus (Fig. S1). First, mutant arnA was amplified and combined with the amplified selectable marker in a second PCR step. The resulting PCR product containing mutated arnA and the selectable marker was transformed into the arnA knockout strain for recombination using the λ Red recombinase plasmid (pKD46). The selectable marker was eliminated using the FLP plasmid (pCP20). For the modification in slyD, the arnA mutant strain was transformed with a PCR product (generated using primers 5F and 5R) containing a selectable marker flanked by homologous overhangs that, after recombination, result in the elimination of the 46-residue C-terminal, histidine-rich segment of SlyD. Again, the selectable marker was later removed using pCP20. Proper genomic integration was confirmed by PCR and sequencing. The RIL plasmid (Agilent Technologies) encoding rare tRNAs was transformed into the final expression strain to improve the expression of our eukaryotic target proteins.
The binding affinity of wild type and mutant ArnA were assessed by immobilizing purified protein onto a 1 ml His-Trap FF column (GE Healthcare) equilibrated in 50 mM potassium phosphate pH 8.0, 300 mM NaCl, and 5 mM beta-mercaptoethanol. Protein was eluted with a linear gradient of 0–150 mM imidazole. The imidazole concentration at the elution peak of each protein was recorded and compared.
Growth analysis was performed at 18, 25 and 37°C for both LOBSTR and the BL21(DE3) strains carrying the same test expression plasmid (See table S2 for a list of all test constructs). Cultures of 1L were grown in LB medium supplemented with 0.4% (w/v) glucose and antibiotic selection at 37°C to OD600 ~0.7. Protein expression was induced with 0.2 mM IPTG 20 minutes after the cultures were shifted to the desired expression temperature. OD600 was measured from the initial synchronization time and until the cells were harvested ~20–22 hours after induction.
To test protein purification, BL21(DE3) and LOBSTR cultures were started at 37°C in LB medium supplemented with 0.4% (w/v) glucose and appropriate antibiotic selection. At OD600 ~0.7, cultures were shifted to 18°C and induced with 0.2 mM IPTG ~20 min later. Cultures were harvested after 18–20 hours. For each strain and construct tested, a total of ~3.5g of cells were resuspended in 50 mL of resuspension buffer (40 mM potassium phosphate pH 8.0, 150 mM NaCl, 40 mM imidazole, and 3mM beta-mercaptoethanol) and lysed with a cell disrupter (Constant Systems). Lysates were cleared for 25 min at 9500×g and the soluble fraction was incubated with 400 µl bed volume of Ni Sepharose 6 Fast Flow (GE Healthcare) resin for 1 hour while stirring at 4°C. The resin was collected and washed with 6 mL of resuspension buffer and eluted with 2 mL of elution buffer (40 mM potassium phosphate pH 8.0, 150 mM NaCl, 250 mM imidazole, and 3 mM beta-mercaptoethanol). Elution fractions were analyzed on a 4–15 % SDS-PAGE gradient gel (Bio-RAD) and stained with Coomassie Blue R250. Purifications using Ni-NTA (Qiagen) and Talon (Clontech) resins were performed using resuspension buffer containing 20 mM or 5 mM imidazole, respectively, following manufacturer’s recommendations.

The efficiency of the biotinylation reaction has been examined by Western blotting (6 (link)) or other enzymatic or ligand-displacement assays (46 (link)), but these approaches are time-consuming and only indirectly allow quantitation. A rapid and easily quantified alternative is to saturate the target protein with streptavidin and study the gel-shift in SDS-PAGE (seeFig. 5). Provided the gel does not get excessively warm during the run, streptavidin will retain its native tetramer structure and remain bound to biotin conjugates under normal SDS-PAGE conditions (16 (link)). A streptavidin monomer (i.e. one biotin binding site) has a calculated ε₂₈₀ of 41,940 M⁻¹ cm⁻¹.

Prepare a PCR tube containing 5 μL of 10 μM biotinylated target protein and add 10 μL of 2× SDS-PAGE buffer.

Heat samples at 95 °C for 5 minutes in a PCR block with a heated lid.

Allow the sample to cool to room temperature and briefly centrifuge.

After this boiling and cooling, add 5 μL of PBS containing a small molar excess (2- to 5-fold) of streptavidin to the samples and incubate at room temperature for 5 minutes (it is advisable to run a control lane of streptavidin without the target protein).

Run samples on an appropriate SDS-PAGE gel (the streptavidin tetramer, running at 50-60 kDa, is clearly visible on 10, 12, 14, 16 % gels) (seeNote 7).

Stain the gel with InstantBlue or Coomassie blue and visualize. If desired, quantify the degree of biotinylation by densitometry, measuring the change in intensity of the relevant protein band with and without addition of streptavidin (seeNote 8). In the lane containing biotinylated protein and streptavidin, the presence of a band corresponding to free streptavidin verifies that streptavidin was indeed provided in excess and so all biotinylated protein will have been bound. Streptavidin may sometimes increase in mobility upon binding to biotin conjugates, according to the size and charge of the biotin conjugate (seeFig. 5).

The proteins and protein complexes (i.e. Mre11-Rad50-Xrs2, Dna2, yeast and human RPA proteins, Sgs1, Top3-Rmi1, Srs2, Fen1) used in this study were expressed in insect, yeast, or E. coli cells and purified as detailed in the Supplementary Information. E. coli SSB protein was purchased (New England BioLabs). For the end resection reactions, linear DNA was 3′ or 5′ labeled with ³²P using standard methods, and the internally ³²P-labeled DNA was generated by PCR. Other ³²P-labeled DNA substrates for testing Sgs1 and Dna2 DNA helicase and Dna2 nuclease activities were prepared as described in the Supplementary Information. The supercoiled φX174 DNA was purchased (New England BioLabs) and its linearization was by digestion with the restriction enzyme StuI. The DNA resection reactions were analyzed in agarose or polyacrylamide gels under denaturing or non-denaturing conditions, followed by phosphorimaging analysis of the dried gel or ethidium bromide treatment of the gel to visualize DNA species. The pulldown experiments to test for protein-protein interactions made use of affinity tags on the indicated proteins. Visualization of proteins was by the staining of SDS polyacrylamide gels with Coomassie Blue or by immunoblotting. Experimental details for the Dna2 nuclease, double Holliday Junction dissolution, DNA helicase, and ATPase assays, and pre-resection of uniformly ³²P-labeled DNA can be found in the Methods section or Supplementary Information. Genetic assays to measure the distribution of recombinant products of the gene conversion and crossover types or the kinetics of the resection of a site-specific DNA double strand break were conducted as described6 (link),7 (link).