{"id":373,"date":"2019-03-03T16:33:12","date_gmt":"2019-03-03T08:33:12","guid":{"rendered":"http:\/\/metabolismcompoundlibrary.com\/?p=373"},"modified":"2022-01-13T18:12:14","modified_gmt":"2022-01-13T09:42:14","slug":"omrb-electrophoresis-detect-dna-dsbs-cells-blm-exposure","status":"publish","type":"post","link":"http:\/\/metabolismcompoundlibrary.com\/index.php\/2019\/03\/03\/omrb-electrophoresis-detect-dna-dsbs-cells-blm-exposure\/","title":{"rendered":"Together with OmrB electrophoresis to detect DNA DSBs in cells after BLM exposure"},"content":{"rendered":"

The RecBCD exonuclease acts on DNA ends generated by DSBs and so we used a recBCD strain to stabilize such ends. Wildtype or recBCD cells were grown in either L broth or glucose or minimal medium and exposed to various concentrations of BLM for 30 min, centrifuged and washed once to remove BLM, and then incubated for a further 30 and 60 min in respective growth medium. Samples of the washed and incubated cell population were removed, embedded in agarose and subjected to pulse field gel electrophoresis. The results are shown in Fig. 6. The wildtype strain, GM7330, Taltirelin<\/a> growing in L broth, shows a dose-dependent increase in low molecular weight DNA after BLM exposure. Compared to the wildtype strain, the recBCD strain growing in L broth shows a dosedependent increased accumulation of low molecular weight DNA after BLM exposure. In glucose minimal medium, however, there is no dose-dependent accumulation of low molecular weight DNA in either the wildtype or recBCD cells. These results indicate that, after BLM exposure, DSBs are detectable in cells growing in broth but not in cells growing in glucose minimal medium. To gain insight on the mechanism of resistance of cells to BLM, total RNA was isolated from bacteria growing in either L broth or glucose minimal medium and challenged or not with 0.7 mM BLM. The microarray data from exponentially growing BLM treated cells was compared to that from untreated cells. The abbreviated results are shown in Table 1 and the complete data are in Tables S1 and S2. Wildtype bacteria exposed to BLM in L broth show a robust SOS regulon induction with the recN gene showing the greatest increase. As expected, there is no induction of SOS genes in the recA strain in either media. In the recA strain growing in L broth and exposed to BLM, there is increased induction of the initiator of chromosome replication protein, DnaA; the ribonucleotide reductase genes, and its associated cofactor ferridoxin gene, yfaE. These results suggest that there is increased initiation at oriC and increased levels of deoxyribonucleoside triphosphates in BLM-stressed recA cells growing in L broth. In glucose minimal medium, SOS induction occurs in wildtype cells exposed to BLM but the fold induction and the number of expressed SOS genes is less than in L broth. The sokC gene encodes a small regulatory RNA that indirectly Folic acid<\/a> blocks translation of the HokC toxic membrane polypeptide. The mltA gene encodes a membrane-bound murein hydrolase and the napA gene encodes a nitrate reductase. The proV gene produces a high affinity glycine transporter and the fdnH gene encodes a subunit for formate hydrogenase N, an integral inner membrane protein. It is not clear how increased expression of any of these non-SOS genes can produce BLM resistance. In the recA bacteria in glucose minimal medium and exposed to BLM, the gene with the largest fold increase is rygA, which encodes a small non-coding regulatory RNA.<\/p>\n","protected":false},"excerpt":{"rendered":"

The RecBCD exonuclease acts on DNA ends generated by DSBs and so we used a recBCD strain to stabilize such ends. Wildtype or recBCD cells were grown in either L broth or glucose or minimal medium and exposed to various concentrations of BLM for 30 min, centrifuged and washed once to remove BLM, and then […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[1],"tags":[],"_links":{"self":[{"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/373"}],"collection":[{"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/comments?post=373"}],"version-history":[{"count":1,"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/373\/revisions"}],"predecessor-version":[{"id":374,"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/posts\/373\/revisions\/374"}],"wp:attachment":[{"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/media?parent=373"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/categories?post=373"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/metabolismcompoundlibrary.com\/index.php\/wp-json\/wp\/v2\/tags?post=373"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}