Lambda dna

Unless otherwise stated, 0N80 end-positioned nucleosomes with and without DNA lesions (140 nM) were incubated with INO80-C complexes (50 nM) in sliding buffer [25 mM HEPES pH8.0, 50 mM NaCl, 5% glycerol, 1 mM TCEP and 2 mM MgCl₂] in a final volume of 10 μl at room temperature. For reactions presented in Figure 1, different concentrations of NaCl were used. Sliding was initiated by adding 1 mM ATP, and the reaction was quenched by adding 5 mM EDTA and 0.2 mg/ml lambda DNA (NEB). Reactions at different time points were collected and resolved on 6% Native-PAGE gels at 4 °C (100 V, 90 min, 1× TBE). Gels were stained with SYBR-GOLD (GoldBio) before imaging on a Typhoon imager (Cytiva). Quantification of the gels was done using ImageJ software version 1.53e. The percentage of fully remodeled nucleosomes was plotted against time using GraphPad (Prism) software.

All ODNs used in this study were ordered from Integrated DNA Technologies, Inc. (Coralville, IA, USA) and dissolved in sterile Milli-Q water to 100 µM for stock. M13mp18 single-stranded DNA (M13 ssDNA, 250 µg/mL), M13mp18 RF I DNA (M13 dsDNA, 100 µg/mL) and Lambda DNA (300 µg/mL) were purchased from New England Biolabs, Inc. (Beverly, MA, USA). Salmon sperm DNA (Sigma-Aldrich, WI, USA) was dissolved in sterile Milli-Q water to 300 µg/mL and used as the substrate for depurination. Nucleotide bases, including adenine, guanine, thymine, cytosine and uracil (Sigma-Aldrich, WI, USA) were used as the standard substances for HPLC analysis. They were dissolved in sterile Milli-Q water to 200 µg/mL, respectively, and diluted to the final concentration of 20 µg/mL in mixed samples.

The 300 and 2000 bp linear DNA fragments were generated by amplifying the first 300/2000 bp of lambda DNA (NEB, Ipswich, USA) by PCR using the forward primer BR215_Cy5_tetO_lambda_fw and the reverse primers BR120_5′BiotinTEG_l300_rev and BR122_5′BiotinTEG_l2000rev (Sigma-Aldrich), respectively. The resulting PCR products were biotinylated and labeled with Cy5 on opposite ends. PCR products were purified and purity and labeling was assessed by gel electrophoresis. SLBs were generated as described under streptavidin-bound membranes using non-labeled streptavidin. After removal of surplus streptavidin from the reaction chamber, reaction buffer was added to a volume of about 50 µl and 6 pmol/2 pmol of the 300 bp/2000 bp long PCR product was added. DNA containing chambers were incubated for 2–3 h, then unbound DNA was removed by gently washing three times with a total of 600 µl reaction buffer. To start self-organization 1 µM MinD with 30% mol EGFP-MinD, 1 µM MinE, and 2.5 mM ATP in a total of 200 µl reaction buffer were added.

Lambda DNA for nuclear extract experiments on the LUMICKS C-trap was purchased from New England Biolabs. Biotin tags was added to the DNA by adding a mix of 6 μg Lambda DNA, 50 μM nucleotide mix (with dATP, dGTP, dTTP and biotinylated dCTP), 15 units of Klenow fragment polymerase (NEB) and 1x concentration of NEB Buffer 2. The reaction was incubated for 30 min at 37°C and then the free nucleotides were removed from solution via ethanol precipitation, with 1 μg/μl glycogen used as a co-precipitant to increase the yield. DNA containing Hx and Cy3 fiducial markers of the damage positions was generated by first treating 1 μg of DNA with the nickase Nt.BspQI (NEB) to generate 10 nicks in Lambda DNA at specific sites. Two of the positions are close together and not resolved in our assay and another is too close to the bead to be observed so only 8 sites are observed. After nicking the DNA, fluorescent nucleotides were incorporated using nick translation for identification of nick sites, using a 40 μM mix of dGTP, dCTP, dITP (deoxyinosine triphosphate, the nucleotide form of hypoxanthine) and Cy3-labeled dUTP, in the presence of 10 units of pol I and 800 ng nicked Lambda DNA.

Genomic (g)DNA was extracted from the seeds/seedlings using the Qiagen DNeasy Plant mini kit. Purified gDNA (600 ng) was used for MethylC-seq library preparation after spiking in 0.5% lambda DNA (N6-methyladenine-free) (New England BioLabs) [87 (link)]. Three biological replicates were conducted per time point. Following bisulfite conversion and purification, the adapter-ligated DNA molecules were sequenced using the Illumina HiSeq 1000, following manufacturers’ instructions. For visualisation in JBrowse we used the methylation plugin (https://github.com/bhofmei/jbplugin-methylation), courtesy of Brigitte Hofmeister. Note for Fig. 1b and Additional file 2: Figure S10, data were shown within the AnnoJ genome browser (http://www.annoj.org/).