Uracil Repair - A Source of DNA Glycosylase Dependent Genome Instability

Uracil DNA glycosylases (UDGs) excise uracil from DNA arising from dUMP misincorporation during replication or from cytosine deamination. Besides functioning in canonical uracil repair, UDGs cooperate with DNA base modifying enzymes to effect mutagenesis or DNA demethylation. Mammalian cells express four UDGs, the functional dissection of which represents a challenge. Here, we used Schizosaccharomyces pombe with only two UDGs, Ung1 and Thp1, as a simpler model to study functional interactions in uracil repair. We show that despite a predominance of Ung1 activity in cell extracts, both UDGs act redundantly against genomic uracil accumulation and mutations from cytosine deamination in cells. Notably, Thp1 but not Ung1-dependent repair is cytotoxic under genomic uracil stress induced by 5-fluorouracil exposure or AID expression. Also, Thp1- but not Ung1-mediated base excision is recombinogenic, accounting for more than 60% of spontaneous mitotic recombination events in a recombination assay. Hence, the qualitative outcome of uracil repair depends on the initiating UDG; while Ung1 shows expected features of a bona-fide DNA repair enzyme, Thp1-initiated repair appears slow and non-productive, implicating a function beyond canonical DNA repair. Given the epigenetic role of mammalian TDGs, we performed transcriptome analyses and identified a possible function of Thp1 in stabilizing gene expression. A cross was performed between an h+ wild-type (WT) and an h- Thp1-deficient (thp1Δ) S. pombe strain. Nine WT (h+) and nine thp1Δ (h+) strains were isolated from the cross and grown on YEL medium. Cells were not frozen prior to RNA extraction. RNA was extracted from all 18 strains. RNA of three strains of each genotype were pooled after RNA extraction and prior to the tiling array. Three of these RNA pools were analyzed on tiling arrays for each genotype (six samples in total). Single probes were attributed to genomic elements. Relative expression was received by normalizing the thp1Δ mean expression value to that of the WT. To check the variability of gene expression, the expression values of all six samples were compared without normalizing to the WT.