title: Excision of uracil from transcribed DNA negatively affects gene expression.
abstract: Uracil is an unavoidable aberrant base in DNA, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires a succession of intermediate products, including an abasic site and a single strand break, before the original DNA structure can be reconstituted. These repair intermediates are harmful for DNA replication and also interfere with transcription under cell-free conditions. However, their relevance for cellular transcription has not been proved. Here we investigated the influence of uracil incorporated into a reporter vector on gene expression in human cells. The expression constructs contained a single uracil opposite an adenine (to mimic dUTP misincorporation during DNA synthesis) or a guanine (imitating a product of spontaneous cytosine deamination). We found no evidence for a direct transcription arrest by uracil in either of the two settings because the vectors containing the base modification exhibited unaltered levels of enhanced GFP reporter gene expression at early times after delivery to cells. However, the gene expression showed a progressive decline during subsequent hours. In the case of U:A pairs, this effect was retarded significantly by knockdown of UNG1/2 but not by knockdown of SMUG1 or thymine-DNA glycosylase uracil-DNA glycosylases, proving that it is base excision by UNG1/2 that perturbs transcription of the affected gene. By contrast, the decline of expression of the U:G constructs was not influenced by either UNG1/2, SMUG1, or thymine-DNA glycosylase knockdown, strongly suggesting that there are substantial mechanistic or kinetic differences between the processing of U:A and U:G lesions in cells.
authors.affiliation: From the Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Bork
authors.initials: B
authors.lastname: Lühnsdorf
authors.affiliation: From the Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, Staudingerweg 5, 55128 Mainz, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Epe
authors.affiliation: From the Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, Staudingerweg 5, 55128 Mainz, Germany khobta@uni-mainz.de.
authors.forename: Andriy
authors.initials: A
authors.lastname: Khobta
mesh_terms: D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D045647:DNA Glycosylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D044502:Thymine DNA Glycosylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014498:Uracil; D004247:DNA; C492570:CCNO protein, human; D045647:DNA Glycosylases; C498095:SMUG1 protein, human; D044502:Thymine DNA Glycosylase; D051981:Uracil-DNA Glycosidase
keywords: Base Excision Repair (BER); DNA Damage Response; DNA Repair; Flow Cytometry; Gene Transcription; Reporter Gene; Transfection; Uracil in DNA; Uracil-DNA Glycosylase