Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

Abstract

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 −/− mice. We found that 5-hydroxymethyluracil accumulated in Smug1 −/− tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 −/− mice did not accumulate uracil in their genome and Ung −/− mice showed slightly elevated uracil levels. Contrastingly, Ung −/− Smug1 −/− mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Uracil moieties in Plasmodium falciparum genomic DNA

“…These two limitations relate to, on the one hand, the set of enzymes capable of recognizing and cleaving uracil from DNA, and on the other hand, to the set of proteins required for the short-patch versus long-patch BER routes. Uracil-DNA glycosylases in diverse organisms include at least four enzyme families (UNG, TDG, SMUG, MBD4) [35,36]. The diversity in these enzymes defines their specific roles and different substrate specificities and underlies the high significance of uracil removal from DNA.…”

SMUG1 Promotes Telomere Maintenance through Telomerase RNA Processing

“…It is possible that the function of SMUG1 in Terc biogenesis contributes to the apparently stronger telomere phenotype in Smug1 À/À mice, but although SMUG1 binds Terc in MEFs ( Figure S1F), Smug1 À/À MEFs did not show consistently reduced Terc levels ( Figure S1G). Therefore, the telomere maintenance defects in MEFs appear to be mainly caused by the loss of SMUG1-dependent BER (Alsøe et al, 2017). Measurements of telomere fragility (Figures 2D, 2E, and S1D) indicated impaired replication of the C-rich telomere strand, which would be expected to contain more uracil lesions.…”

“…We previously observed that overexpression of a SMUG1 mutant unable to interact with DKC1 (E29R/E33R) affected DKC1 localization in HeLa cells (Jobert et al, 2013). To confirm that disruption of the SMUG1/DKC1 interaction surface perturbs proper localization of DKC1, we repeated these experiments in Smug1 À/À mouse embryonic fibroblasts (MEFs) (Alsøe et al, 2017). To exclude any bias originating from possible small differences in cell-cycle distribution, we scored the ring-shaped structures formed by DKC1 (DKC1 circles) during S phase (Lee et al, 2014).…”

“…Animal maintenance, mouse handling and experimental procedures were performed in accordance to institutional guidelines and procedures approved by the Animal Experimentation Administration in Norwegian Food Safety Authority (NFDA). The generation of Smug1 À/À C57BL/6J mouse model was described previously (Alsøe et al, 2017).…”

“…Primary and transformed wild-type and Smug1 À/À mouse embryonic fibroblasts (MEFs) were generated from the gene-targeted Smug1 À/À , Smug1 tm1Hln , C57BL/6J mouse model previously established in our lab (Alsøe et al, 2017). Timed matings were set up between either wild-type or Smug1 À/À mice born from heterozygous parents in order to obtain wild-type and Smug1 À/À embryos.…”