A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.
Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity.
Hasegawa
Mayu
M
Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
Iwai
Shigenori
S
Kuraoka
Isao
I
D001681:Biological Assay; D001976:Bromouracil / Q000031:analogs & derivatives; D002460:Cell Line; D003057:Cockayne Syndrome / Q000235:genetics; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D012313:RNA / Q000096:biosynthesis*; D012318:RNA Polymerase I / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000235:genetics
D001976:Bromouracil; D012313:RNA; D012319:RNA Polymerase II; D012318:RNA Polymerase I; C006824:5-bromouridine; D014529:Uridine