The contribution of different uracil-DNA glycosylases to removal of uracil from DNA in different mouse organs - the significance of sequence context and proliferative status
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Uracil is a non-canonical base in DNA that can arise through misincorporation of dUMP instead of dTMP during replication or from cytosine deamination. Uracil in DNA can be removed by the four different DNA glycosylases UNG, SMUG1, TDG or MBD4 as the first step in base excision repair. These glycosylases have different catalytic efficiencies, different substrate preferences and different expression patterns. We wanted to elucidate the contribution of the different DNA glycosylases in removal of uracil, using protein extracts from mouse brain, small intestine, kidney, liver, lung and muscle. Importantly, UNG+/+ and UNG-/- mice were available for these studies. Furthermore, we used the UNG-specific inhibitor Ugi to inhibit UNG-activity and an anti-SMUG1 antibody PSM1 to inhibit SMUG1 activity. In addition, we used a combination of Ugi and PSM1 to inhibit both UNG and SMUG1 activities. To investigate the contribution of the DNA glycosylases in different sequence contexts, we used uracil-containing DNA substrates with uracil in a U:G mismatch, a U:A pair, or in single stranded DNA. We found that UNG was by far the most important DNA glycosylase removing uracil from U:A pairs and from single-stranded DNA, even in extracts from non-proliferative tissues such as brain and muscle. However, we found that UNG and SMUG1 are almost equally active in removal of uracil from a U:G context in extracts from non-proliferative tissues. In contrast, SMUG1 contributed very little to the removal of uracil from small intestines, which presumably contain a higher fraction of rapidly dividing cells. Furthermore, we detected essentially no residual uracil-DNA glycosylase activity when both UNG and SMUG1 were specifically inhibited, indicating that TDG and MBD4 do not contribute measurable activity in any of the organs investigated under the conditions used here. Possibly these DNA glycosylases can only contribute in sequence patterns that have not been investigated here. We also carried out a preliminary study on the possible correlation between genomic uracil contents (measured by mass spectrometry) and uracil excision activities, but failed to observe a significant positive correlation or inverse correlation. In conclusion, UNG is the dominant uracil-DNA glycosylase in proliferating tissues in removal of uracil from a U:G context, whereas in non-proliferating tissues the contribution of UNG and SMUG1 is essentially similar. Furthermore, UNG is the only glycosylase removing uracil from U:A and single stranded DNA contexts.