GSE159048
Mycobacteria excises DNA damage in 12- or 13-nucleotide-long oligomers by prokaryotic-type dual incisions and performs transcription-coupled repair
In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs) are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual incision patterns have been discovered: prokaryotic type which removes the damage in 11-13 nucleotide-long oligomers and eukaryotic type which removes the damage in 24-32 nucleotide-long oligomers. However, a recent study reported that the UvrC protein of Mycobacterium tuberculosis removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nucleotides from the 3' end and 9 nucleotides from the 5' end flanking the damage. To test this model we used the in vivo excision assay and the XR-seq genome-wide repair mapping method developed in our lab to determine the repair pattern and genome-wide repair map of M. smegmatis. We find that M. smegmatis, which possesses homologues of the E. coli UvrA, B, and C genes, removes CPDs from the genome in a manner identical to the prokaryotic pattern by incising 7 nucleotides 5' and 3 or 4 nucleotides 3' to the photoproduct. Overall design: We apply XR-seq in Mycolicibacterium smegmatis MC2 155, the cells were grown with shaking at 37 °C in Luria Broth containing 0.5% (w/vol) Tween 80. Exponentially growing cells were transferred to R150 tissue culture dishes in volumes of 15 mL and were irradiated at an OD600 of ~0.6 at room temperature with 100 J/m2. Dishes were then incubated for 10 min at room temperature, and then cells were harvested, chilled, and maintained on ice. Excision products were then isolated using a modified Hirt procedure that employs shearing to assist cell lysis.  Cells were pelleted at 4 °C, resuspended in ice-cold TE [Tris (10 mM)-EDTA (1 mM) (pH 8.0)], transferred to ice cold Eppendorf tubes, and pelleted at 4 °C, and the supernatants were removed. Pellets were then resuspended with 360 µL of ice-cold TE. Then, 50 µL of room temperature 10% (wt/vol) SDS was added, and cells were lysed by passage through a 26G needle 40 times followed by incubation at room temperature for 20 min. A 120-µL volume of room temperature NaCl (5 M) was then added, tubes were gently mixed, and suspensions were incubated at 4 °C overnight. After centrifuging at high speed for 1 h in a microfuge at 4 °C, supernatants (about 480 µL each) were taken, and each was mixed with and incubated with 12 µL of RNaseA (R4642; Sigma) for 1 h at 37 °C, and then with 12 µL of proteinase K (P8107S; NEB) for 1.5 h at 60 °C. Samples were then extracted twice with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. Samples were immunoprecipitated with an anti-CPD antibody and then ligated to adapters and processed for sequencing (XR-seq). XR-seq libraries were pooled and then sequenced on the HiSeq 2500 Rapid Run platform at the UNC-Chapel Hill High-Throughput Sequencing Facility. Analysis pipeline is available on https://github.com/yanyanyangunc/DNA-Damage-Repair-Circadian-Clock/tree/master/Mycobacterium_smegmatis_str_mc2_155