Phosphorimager

20 nM 5′ end ³²P-labeled ssDNA (GTTACGGTTACAGGTTACG) was mixed with Pot1 proteins of specified concentrations in 20 μl reaction buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 5 mM DTT, 2 mM MgCl₂, 10% glycerol). The mixtures were then incubated at 4°C for 30 min. The protein-ssDNA complex were resolved by 7% non-denaturing polyacrylamide gel and imaged with a Bio-Rad phosphorimager.

Genomic DNA preparation and physical analysis of recombination were performed as described previously (19 (link),35 (link)–36 (link)). For 2D gel electrophoresis, genomic DNA was digested with 80 units of XhoI enzyme, and loaded onto 0.4% SeaKem Gold agarose gel in Tris-Borate-EDTA (TBE) buffer, at ∼1 V/cm for 21 h. Gels were stained with 0.5 μg/ml ethidium bromide (EtBr) for 30 min, and afterward the lanes containing DNA of interest were cut out to prepare gel slices. Following this, SeaKem LE agarose (0.8%) with 0.5 μg/ml EtBr was added to the 2D gel tray. The second gel electrophoresis was carried out at ∼6 V/cm, for 6 h at 4°C. Southern blot analysis was performed using ³²P-dCTP–labeled radioactive nucleotides, prepared with the Random Primer labeling kit (Agilent Technologies, USA). DNA hybridization signals were quantified using a Bio-Rad Phosphorimager and Quantify One software (Bio-Rad, USA). Detection of DSBs in the rad50S background was performed as described previously (19 (link)).

For RNA extractions cells were grown at 30°C till OD_{600 nm} was 0.6 and then continued at 30°C or shifted to 37°C for 90 min before harvesting. Total cellular RNA was isolated using the acidic hot phenol method as previously described (Arimbasseri and Bhargava 2008 (link)). Typically, 600–1000 ng RNA was used for anaysis on a 2.8% agarose gel, run in 0.5× TBE buffer. Bands were visualized under UV exposure after ethidium bromide staining. The Pol III-transcribed total RNA was measured by the Real-Time PCR method using 5 µg of RNA for cDNA synthesis (Kumar and Bhargava 2013 (link)). For all measurements made, averages from at least three biological replicates and scatter have been plotted. For northern blotting (Karkusiewicz et al. 2011 (link); Vernekar and Bhargava 2015 (link)), 15 µg of RNA was used per sample. Pol II-transcribed U4 snRNA gene was used as positive control in the northern blots, which were sequentially probed with the 5′ end-labeled gene-specific oligos and visualized in a Bio-Rad PhosphorImager. RNA levels were also estimated by the tRNA-HySeq method, essentially as described previously (Arimbasseri et al. 2015 (link)) and detailed previously (Bhalla et al. 2019b (link)).