Escherichia coli rnase hii

The assay was performed as described in details in (45 (link)). Briefly, genomic DNA was isolated using Y-DER (Thermo Fisher Scientific, Waltham, Massachusetts USA) extraction kit according to manufacturer's instruction and treated with Escherichia coli RNase HII (NEB, Ipswich, Massachusetts USA), which introduces nicks at every ribonucleotide-containing site. The nicks were then used to radioactively label the site with DNA Polymerase I (NEB, Ipswich, MA, USA) in the presence of unlabeled dA/-dT/-dGTP and α-³²P-dCTP (Perkin Elmer, Waltham, MA, USA). Labeled genomic DNA was separated by agarose gel electrophoresis in the presence of ethidium bromide, imaged under UV light and quantified with ImageLab software (Bio-Rad). The gel was successively dried, and the radioactive signal was detected by autoradiography using a Typhoon FLA 7000 (GE Healthcare Life Sciences, Marlborough, MA, USA) and quantified with ImageQuant software (GE Healthcare Life Sciences, Marlborough, MA, USA). The radioactive signal of each sample was normalized on total genomic DNA measured by ethidium bromide staining. The ratio between in-vitro RNHII-treated and the untreated sample was expressed as fold change respect to the control sample. The mean of four different experiments ± SEM is reported in the figure.

Genomic DNA was purified using Qiagen Genomic-Tip and Qiagen Genomic DNA buffer set and was digested with 0.5 U Escherichia coli RNase HII (New England Biolabs) in 50 µl at 37°C for 2.5 h. After precipitation in 0.3 m sodium acetate, pH 7, and ethanol, DNA was resuspended in TE 0, 1%. Nick translation was performed using 5 U of E. coli DNA polymerase I (New England Biolabs), 20 µm of unlabeled dA/-T/-GTP, and 5 µC α³²PdCTP in a final volume of 20 µl. The reaction was incubated at 16°C for 30 min. Labeled DNA was separated from unincorporated nucleotides by electrophoresis on a 1% TAE agarose gel. After drying, the radioactive gel was analyzed using a Typhoon. The relevant ³²P-signal of each sample was normalized on total genomic DNA measured by ethidium bromide staining. The ratio between E. coli RNHII-treated and untreated sample was expressed as fold change respect to the control sample.