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.