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Selective modulation of the functions of a conserved DNA motor by a histone fold complex

Budding yeast Mph1 helicase and its orthologs drive multiple DNA transactions. Here, Xue et al. show that the conserved histone fold MHF complex promotes Mph1-mediated repair of damaged replication forks but does not influence the outcome of DNA double-strand break repair.


Supplemental Methods
Detailed methods for protein purification, analyses of protein-protein and protein-DNA interactions, Mph1 activity assays, 2D gel analyses, DSB repair and crossover assays.

Figure S3. scMHF alone does not influence Mph1 fork regression and branch migration activities in vitro. (A)
The effect of scMHF on replication fork regression by Mph1 was examined. Mph1 (2 nM) and scMHF (4 nM) were incubated with MRF (15 nM) at 30°C for the indicated times.

(B)
The effect of scMHF on DNA branch migration by Mph1 was examined. Mph1 (2 nM) and scMHF (4 nM) were incubated with MHJ (15 nM) at 30°C for the indicated times. The asterisk denotes the 32 P label. The results were quantified and plotted. Error bars represent standard deviation from three independent experiments. (C-D) scMHF does not affect the processing of a sigma structure by Mph1. (C) Schematic of the sigma structure used to assess replication fork regression and HJ branch migration activities.
Regression of the fork structure by Mph1 yields a HJ, which can further branch migrate over 2.9 kb to generate a radio-labeled linear product and an unlabeled circular one. The asterisk denotes the 32 P label. (D) Mph1 (1 nM) and scMHF (2 nM) were incubated with the sigma structure (0.5 nM) and then analyzed after the indicated incubation times at 30°C. The results were quantified and plotted. Error bars represent standard deviation from three independent experiments. (B-C) scMHF and Smc5 have no effect on Mph1's D-loop dissociation activity. Assays were performed as described previously (Prakash et al., 2009;Xue et al., 2014  and solvent-exposed positively charged residues in both hMHF subunits, features that are germane for DNA engagement by hMHF. PDB 4NDY for hMHF in complex with dsDNA and 3V9R for scMHF were used to generate the vacuum electrostatics (Singh et al., 2010;Tao et al., 2012;Yan et al., 2010). Positively and negatively charged residues are colored blue and red, respectively.

Expression and purification of Mph1 and Smc5
Published methods (Prakash et al., 2009;Xue et al., 2014;Zheng et al., 2011) were followed. Briefly, Smc5 and mph1 (754-993) were expressed in E. coli, whereas Hi5 insect cells and recombinant baculoviruses were used for expressing Mph1 and mph1 (1-754). Proteins were purified to near homogeneity by the combination of affinity chromatography, ion exchange, and size exclusion chromatography (Prakash et al., 2009;Xue et al., 2014;Zheng et al., 2011) Expression and purification of (His) 6 -tagged scMHF and mutant The budding yeast MHF1 and MHF2 genes were introduced into the pETDuet vector with a (His) 6 tag sequence at the N-terminus of Mhf2. E .coli BL21:DE3 Rosetta cells harboring pETDuet-Mhf1/(His) 6 -Mhf2 were grown at 37°C to OD600 = 0.8, and protein expression was induced by the addition of 0.2 mM IPTG and incubation at 16°C for 16 h. Cells were harvested by centrifugation and all the subsequent steps were carried out at 0 to 4°C. For lysate preparation, a cell pellet (10 g, from 2 L of culture) was suspended in 50 ml K buffer (20 mM KH 2 PO 4 , pH 7.4, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT) with 150 mM KCl and 5 µg/ml each of aprotinin, chymostatin, leupeptin and pepstatin, and then subject to sonication (three 1 min pulses). The crude cell lysate was clarified by ultracentrifugation (100,000Xg for 90 min) and then mixed gently with 2 ml of Nickel-NTA resin (Qiagen) for 2 h. The resin was washed sequentially with 3 x 30 ml K buffer containing 300 mM KCl, 3 x 30 ml K buffer containing 150 mM KCl, and 20 ml K buffer containing 150 mM KCl and 20 mM imidazole. The tagged scMHF was eluted with 10 ml K buffer containing 150 mM KCl and 200 mM imidazole and concentrated to 0.5 ml (Amicon 10K concentrator, Millipore) and fractionated in a Superdex 200 gel filtration column (24 ml) in K buffer containing 150 mM KCl. Fractions containing highly purified scMHF (10 mg protein) were pooled, concentrated to 8 mg/ml, and stored in small aliquots at -80°C. The scMHF mutant (mhf1-R36A/L44A/S54A, mhf2-E70A/L75E) was purified using the same procedure with a similar yield.

Expression and purification of GST-and (His) 6 double-tagged scMHF
The MHF1 and MHF2 genes were introduced into the pGex-BICIS vector to construct pGex-BICIS-GST-Mhf2/(His) 6 -Mhf1. Cell growth, protein induction, and cell lysate preparation were as above. The clarified lysate was incubated with 2 ml glutathione Sepharose 4 Fast Flow resin (GE Healthcare) for 1 h. The glutathione resin was washed with 3 x 30 ml of K buffer containing 150 mM KCl, 5 mM ATP, and 10 mM MgCl 2 and 20 ml of K buffer containing 150 mM KCl. The bound GST-Mhf2/(His) 6 -Mhf1 was eluted in K buffer containing 150 mM KCl and 20 mM reduced glutathione. The eluate was mixed gently with 2 ml of Nickel-NTA resin (Qiagen) for 2 h. The resin was washed sequentially with 3 x 30 ml K buffer containing 300 mM KCl, 3 x 30 ml K buffer containing 150 mM KCl, and 20 ml K buffer containing 150 mM KCl and 20 mM imidazole. Bound scMHF was eluted with 10 ml K buffer containing 150 mM KCl and 200 mM imidazole, concentrated to 0.5 ml (Amicon 10K concentrator, Millipore), and fractionated in a Superdex 200 gel filtration column (24 ml) in K buffer containing 150 mM KCl. Fractions containing highly purified, doubly-tagged scMHF (~5 mg protein) were pooled, concentrated to 5 mg/ml, and stored in small aliquots at -80°C.

Expression and purification of untagged scMHF
Untagged MHF1 and MHF2 genes were introduced into the pRSFDuet vector. Cell growth, protein induction, and cell lysate extract preparation were performed as above. The clarified lysate was fractionated in a 5 ml Q Sepharose column with a 100 ml gradient of 150-600 mM KCl in K buffer. Fractions containing the scMHF peak were pooled (~300 mM KCl) and applied onto a 1 ml column of hydroxyapatite (BioRad), which was developed with a 30 ml gradient of 0-400 mM KH 2 PO 4 in K buffer. The scMHF peak fractions were pooled (~130 mM KH 2 PO 4 ), diluted with an equal volume of K buffer, and then fractionated in a 1 ml DEAE (Pharmacia Biotech) column with a 30 ml gradient of 150-600 mM KCl in K buffer. The scMHF peak fractions were pooled (~240 mM KCl), diluted with an equal volume of K buffer, and further fractionated in a 1 ml Mono Q column with a 30 ml gradient of 150-600 mM KCl in K buffer. The scMHF peak fractions were pooled (~300 mM KCl), concentrated to 0.5 ml, and fractionated in a Superdex 200 gel filtration column (24 ml) in K buffer containing 150 mM KCl. Fractions containing highly purified scMHF (1 mg protein) were pooled, concentrated to 2 mg/ml, and stored in small aliquots at -80°C.

Co-immunoprecipitation in yeast
Yeast strains containing chromosomally tagged proteins were grown to mid-log phase and harvested. Yeast cells were lysed in TBT buffer (20 mM HEPES, pH 7.4, 110 mM potassium acetate, 2 mM MgCl 2 , 0.1% Tween-20) supplemented with 100-300 mM NaCl, 0.5% Tween-20, 1 mM dithiothreitol (DTT), protease inhibitors (pepstatin and phenyl-methylsulfonyl fluoride or PMSF) and DNase I with glass beads by vortexing (MP Biomedicals). Lysates were incubated with either agarose (Calbiochem) or sepharose (Pierce) beads conjugated with Protein A or G and indicated antibodies on a rotating wheel for 2-4 hours at 4˚C to immunoprecipitate tagged proteins. After stringent washing with lysis buffer (4-7 times), the bound proteins were eluted in the SDS-PAGE loading dye and analyzed by Western Blotting using indicated antibodies.

2D gel analysis
Experiments were performed as described previously (Choi et al., 2010). In brief, cells were synchronized in G2 by adding nocodazole to the final concentration of 10 mg/ml together with DMSO at 1% for 2.5 h. Cells were then released from nocodazole arrest into YPD medium containing MMS at a final concentration of 0.033% at 30°C. At the indicated times after release, cells were collected for fluorescence-activated cell sorting (FACS) analysis and purification of DNA intermediates. The DNA samples were digested with HindIII and EcoRV and separated by 2D gel electrophoresis followed by Southern blotting with a probe against ARS305.

Measurements of crossover frequency and DSB repair assays
This method is described in (Prakash et al., 2009). In brief, CO frequency was determined as a ratio of the intensity of the band corresponding to gene conversion with crossover to the intensity of bands corresponding to gene conversion both with and without crossover. We measured the frequency of crossovers among the recombination products 8 h after HO induction. DNA isolated from cells was digested with EcoRI enzyme and separated on 0.8% agarose gel. DNA was transferred to Nylon+ membrane and hybridized with a MATa probe corresponding to 200 bp on each side of HO cut site. To calculate kinetics of repair we normalized the DNA amount using DNA probe specific for APA1 gene.