Excision of uracil from transcribed DNA negatively affects the gene expression [DNA and Chromosomes]

June 20th, 2014 by Luhnsdorf, B., Epe, B., Khobta, A.

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 genome requires a succession of intermediate products, including abasic site and 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 was unproven. Here, we investigated the influence of uracil incorporated into a reporter vector on the gene expression in human cells. The expression constructs contained 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, since the vectors containing the base modification exhibited unaltered levels of the EGFP2 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 significantly retarded by knockdown of UNG1/2, but not by the knockdown of SMUG1 or TDG uracil-DNA glycosylases, proving that it is base excision by UNG1/2 to perturb transcription of the affected gene. By contrast, the decline of expression of the U:G constructs was not influenced by either of the UNG1/2, SMUG1, or TDG knockdowns, strongly suggesting that there are substantial mechanistic or kinetic differences between the processing of U:A and U:G lesions in cells.