Supplemental Information - Genes & · PDF file 2009-01-07 · 1 Supplemental...

Click here to load reader

  • date post

    27-Jan-2020
  • Category

    Documents

  • view

    1
  • download

    0

Embed Size (px)

Transcript of Supplemental Information - Genes & · PDF file 2009-01-07 · 1 Supplemental...

  • 1

    Supplemental Information

    Supplemental material and methods

    ATPase assay

    Mph1 (35 nM) or mph1 D209N (100 nM) was incubated at 30oC with 1.5 mM [γ-32P]

    ATP and φX174 circular (+) strand DNA (25 µM nucleotides) in 10 µl of buffer A (30

    mM Tris-HCl, pH 7.5, 15 mM KCl, 2.5 mM MgCl2, 1 mM DTT, and 100 µg/ml BSA).

    At the indicated times, a 2 µl aliquot of the reaction was removed and mixed with an

    equal volume of 500 mM EDTA to halt ATP hydrolysis. The reaction mixtures were

    resolved by thin layer chromatography in a polyethyleneimine cellulose sheet (Petukhova

    et al., 1998) and the level of 32Pi released was determined by phosphorimaging analysis.

    Topoisomerase-I-linked assay to gauge Rad51-ssDNA nucleoprotein filament

    dissociation

    The assay was conducted at 37oC in buffer R containing 2 mM ATP, 2.5 mM MgCl2, 50

    mM KCl, and an ATP-regenerating system. Rad51 (2 µM) was incubated with φX174

    circular (+) strand DNA (10 µM nucleotides) for 5 min before adding the indicated

    concentration of Mph1 or Srs2 and RPA (200 nM). Following a 3-min incubation,

    topologically relaxed φX174 DNA (12.5 µM nucleotides) and 2.5 U calf thymus

    topoisomerase I (Invitrogen) were incorporated and, after an 8-min incubation, the

    reaction was deproteinized by treatment with SDS (0.5%) and proteinase K (0.5 mg/ml)

    for 3 min. The reaction mixtures were resolved in a 0.9% agarose gel run in TAE buffer

    and the DNA species were stained with ethidium bromide.

  • 2

    Electron microscopy

    All the steps were carried out at 37oC and buffer R containing 2 mM ATP, 2.5 mM

    MgCl2, 50 mM KCl, and an ATP-regenerating system but no BSA. To assemble

    nucleoprotein filaments, Rad51 (2.4 µM) was incubated with the 150-mer

    Oligonucleotide E (7.2 µM nucleotides) for 5 min and then with the indicated

    concentration of Mph1 or Srs2 and RPA (240 nM) for 5 min, as described for the

    Topoisomerase I-linked assay above. The reaction mixtures were diluted 8-fold with

    buffer R without BSA and a 4 µl aliquot was applied to 400-mesh grids coated with

    carbon film and which had previously been glow-discharged in air. After staining for 30s

    with 2% uranyl acetate, the samples were examined in a Tecnai 12 Biotwin electron

    microscope (FEI company) equipped with a tungsten filament at 100 keV. Digital

    images were captured with a Morada (Olympus Soft Imaging Solutions) charge-coupled

    device camera at a nominal magnification of X 87,000.

  • 3

    Supplemental Figures

    Supplemental Figure 1. Analysis of crossover frequency in rmi1Δ and mph1Δ mutants. (A) Frequency of crossover product in ectopic recombination in wild type cells (tGI354) and indicated mutant cells (tGI370, tGI853 and tGI880). (B) Scheme representing HO induced allelic recombination between two homologous chromosomes III used to verify crossover frequency and repair efficiency. The wild type diploid cells (cross of JKM139 and AM476) and mph1∆/mph1∆ diploid cells (cross of tGI773 and tGI828) were plated on YEP-galactose plates, and once the colonies formed, they were replica plated on thr- and ura- drop- out plates. The MATa sequence is cut with HO nuclease while the uncleavable MATα-inc allele serves as donor for recombination. Reciprocally sectored colonies on thr- and ura- drop-out plates indicate the number of crossovers. Frequency of crossover products in wild type and mph1∆/mph1∆ homozygote diploid cell is shown in the table. The HO cut chromosome is marked on both ends with the ADE1 marker. To estimate the efficiency of repair we followed the frequency of ADE1 and THR4 markers maintenance after plating cells on YEP-galactose. Cells that fail to repair the DSB lose the ADE1/THR4-marked chromosome. The frequency of spontaneous ADE1 and THR4 marker loss was estimated on YEPD plates and was below 1% (not shown).

    Prakash_Sup Fig1

  • 4

    Supplementary Figure 2. Binding and unwinding of DNA bubble and HJ by Mph1. (A) The oligonucleotides used to create the DNA substrates are indicated,and the length of the DNA regions in each substrate is indicated in red. (B) The Mph1 protein (10, 50, 150 and 200 nM in lanes 2 to 7) was examined for its ability to bind the DNA Bubble (50 nM) (I) or HJ (50 nM) (II) versus the 5’ D-loop substrate (50 nM), as in Figure 4B. The reaction blank (Bl) was run in lane 1. Treatment of the nucleoprotein complex of Mph1 and DNA substrate with SDS and proteinase K (SDS/PK) resulted in substrate release. (C, D) The ability of Mph1 to unwind the Bubble (50 nM) (C) or HJ (50 nM) (D) substrates was examined as in Figure 4C. The heat-denatured substrate (HD) and the reaction blank (Bl) were run in lane 1 and lane 2, respectively.

    Prakash_Sup Fig2

  • 5

    Supplementary Figure 3. D-loop binding by Mph1. (A) Mph1 (50 nM) was co- incubated with the 5’ D-loop (50 nM) and DNA bubble (50 nM) at varying KCl concentrations (lanes 2 to 6), as in Figure 4B. The reaction blank (Bl) was run in lane 1. The results were plotted. (B) Mph1 (10 to 200 nM; lanes 2 to 7) was incubated with D- loop substrates (50 nM) without (panel III) or with a 5’ tail (panel I) or a 3’ tail (panel II). The reaction blank was run in lane 1, as in Figure 4B. The results from these experiments were plotted in panel IV. In the above reactions, treatment of the nucleoprotein complexes with SDS and proteinase K (SDS/PK) resulted in substrate release.

    Prakash_Sup Fig3

  • 6

    Supplementary Figure 4. Purification and characterization of mph1 D209N. (A) Sequence of the Walker type B motif in Mph1 and the mutant allele D209N. (B) Purified mph1 D209N protein (1 µg in lane 2) was analyzed alongside the wild type protein (1 µg in lane 1) by 7.5% SDS-PAGE and Coomassie Blue staining. (C) Purified Mph1 and mph1 D209N were examined for ATPase activity. (D) The ability of Mph1 (20 and 40 nM) and mph1 D209N (40 and 80 nM) to unwind DNA was examined using the 5’ D-loop substrate (50nM), as in Figure 4C. The heat-denatured substrate (HD) was run in lane 1 and the reaction blank (Bl) was run in lane 2. (E) The mph1 D209N protein (10, 20, 50, 100 and 200 nM in lanes 2 to 7) was examined for its ability to bind the 5’ D-loop substrate (50 nM), as in Figure 4B. The reaction blank (Bl) was run in lane 1. Treatment of the nucleoprotein complex with SDS and proteinase K (SDS/PK) resulted in substrate release.

  • 7

    Supplementary Figure 5. Mph1 does not affect the stability of the Rad51-ssDNA nucleoprotein filament. (A) Schematic (top) and results (bottom) of the topoisomerase-I-linked assay to gauge the helicase-mediated disruption of Rad51-ssDNA nucleoprotein filaments assembled with φX174 (+) strand. The concentration of Srs2 was 50nM and of Mph1 was either 50 nM (lane 7) or 100 nM (lanes 5 and 8). (B) EM analysis of the effect of Mph1 (100 nM) and Srs2 (100 nM) on Rad51 filaments assembled with Oligonucleotide E. The proportions of RPA-ssDNA complex (I) and Rad51-ssDNA nucleoprotein filament (II) were determined for the three reactions (≥900 nucleoprotein complexes were sampled in each case) and the results were plotted (III).

    Prakash_Sup Fig5

  • 8

    Supplementary Figure 6. Activity analysis of BLM, WRN, and RECQ1. The ability of Mph1 (40 nM), RecQ1 (20 nM), BLM (20 nM) and WRN (20 nM) to unwind the HJ substrate was examined as in Figure 4C. The heat-denatured (HD) substrate was run in lane 1 and the reaction blank (Bl) was run in lane 2. .

    Prakash_Sup Fig6

  • 9

    Supplemental Table 1. List of strains used in this study. Strain Parental

    strain Genotype Source

    tGI354 DELho DELhml::ADE1 MATa-inc DELhmr::ADE1 ade1 leu2-3,112 lys5 trp1::hisG ura3-52 ade3::GAL::HO arg5,6::GAL::MATa

    Ira et al., 2003

    tGI772 tGI354 mph1::KanMX this study tGI840 tGI354 rad27::URA3 this study yAP081 tGI354 csm3::KanMX this study yAP083 tGI354 hex3::KanMX this study yAP085 tGI354 hst3::KanMX this study yAP089 tGI354 ctk1::KanMX this study tGI858 tGI354 chl1::KanMX this study tGI770 tGI354 mgs1::KanMX this study tGI789 tGI354 esc2::KanMX this study tGI791 tGI354 rtt106::KanMX this study tGI793 tGI354 mms1::KanMX this study tGI794 tGI354 rtt107::KanMX this study QW214 tGI354 mrc1::KanMX this study QW156 tGI354 rrm3::KanMX this study yAP091 tGI354 elg::KanMX this study tGI370 tGI354 sgs1::KanMX Ira et al., 2003 tGI383 tGI354 srs2::LEU2 Ira et al., 2003 tGI853 tGI354 rmi1::KanMX-lox this study tGI880 tGI354 rmi1::KanMX-lox sgs1::KanMX this study tGI787 tGI354 sgs1::KanMX mph1::KanMX this study YDS065 tGI354 mph1::KanMX srs2::LEU2 this study tGI835 tGI354 mph1::KanMX rad51::URA3

    GAL::RAD51::LEU2 this study

    tGI548 tGI354 srs2::TRP1 rad51::URA3 GAL10::RAD51::LEU2

    Ira et al., 2003

    tGI542 tGI354 sgs1::KanMX rad51::URA3 GAL10::RAD51::LEU2

    Ira et al., 2003

    tGI555 tGI354 rad51::URA3 GAL10::RAD51::LEU2 Ira et al., 2003 tGI784 tGI354 mph1::KanMX srs2::TRP1 rad51::URA3

    GAL10-RAD51-LEU2 this study

    tGI836 tGI354 srs2::TRP1 sgs1::KanMX mph1::KanMX rad51::URA3 GAL::RAD51::LEU2

    this study

    JKM139 MATa hml::ADE1 hmr::ADE1 ade1 leu2-3,112 lys5 trp1::hisG ura3-52 ade3::GAL10::HO

    Lee et al., 1998

    AM476 MATalpha-inc ade1 met13 ura3 leu2-3,112 trp1 thr4::URA3

    Ira et al., 2003

    tGI828