*4.3.4 MBD4*

Methyl-CpG Binding Domain 4 DNA glycosylase (MBD4) is a 66 kDa protein that is predominantly found in the nucleus of the cell. While UNG, SMUG1 and TDG have a similar structure to one another, MBD4 has a N-terminal methyl-binding domain (MBD) which is connected to the C-terminal glycosylase domain that, additionally, has a different structural fold to the other 3 UDGs [85]. However, similar to TDG, MBD4 is able to remove U, T, 5-hmU thymine glycol halogenated pyrimidines and εC

when they are paired with G, and MBD4 is not regulated by the cell cycle, the same as SMUG1 [69]. SMUG1's DNA glycosylase activity might predominantly occur near CpG sites since mice with MBD4 knocked out had an increase in C→T transition mutations at CpG sites (DNA methylation sites) [86, 87]. MBD4 has also been reported to have additional roles in apoptosis, transcriptional regulation and active demethylation [82].

## **4.4 UNG vs. SMUG1 vs. mismatch repair**

While UNG seems to be the predominant UDG in removing genomic U, it does not appear to be essential in the overall process. SMUG1 knockout mice's organs had no increase in genomic U, while UNG knockout mice's organs had a 1.9–2.2-fold increase [75]. However, a UNG/SMUG1 double knockout a much greater increase, especially in the liver (25-fold increase), suggesting that while SMUG1 cannot fully compensate for UNG loss it is able to act as a backup [75]. Surprisingly, mice and humans deficient in UNG induce issues related to immunity like problems with CSR and inducing lymphoid hyperplasia [88–91]; however, UNG/SMUG1 single and double knockout mice do not have reduced one-year survival rates [72]. This would suggest that cells can tolerate U, to a certain level at least, and that the major issues that arise from UNG loss seem to be immune-related, likely due to its importance in the adaptive immune system (as discussed in Section 4.2). Paradoxically, cell death induced by too high a levels of genomic U might be induced by the cell's own DNA repair mechanism that induces ssDNA breaks that could lead to dsDNA breaks that are cytotoxic (similar to the mechanism of CSR), in a cancer setting at least [92]. If this is the case, then that would indicate a significant role for UNG/SMUG1 in inducing the cell death in this setting, rather than the high levels of genomic U.

While UNG/SMUG1 double knockout does not reduce one-year survival rates, a triple knockout including MutS homolog 2 (MSH2), a critical enzyme in the DNA repair mechanism termed Mismatch Repair (MMR) severely reduced survival rates when compared to mice with a single MSH2 knockout [72]. This could suggest that while UNG and SMUG1 are complementary to each other's function in removing genomic U, MMR might act as a last resort when the two proteins are lost; however, it is worth mentioning that MSH2 loss alone significantly decreased one-year survival by itself [72]. Furthermore, UNG/SMUG1/MSH2 triple knockout mice also had an increased chance of cancer development (compared to other knockout combinations), which was mostly lymphoma (maybe due to high C deaminase activity in immune cells via AID), suggesting that these three proteins are key to maintaining genomic stability, potentially via the removal of genomic U [72].

Overall, one could hypothesis that high levels of genomic U, in the acute setting, could lead to cell death induced by the DNA repair machinery not being able repair the damage it inflicts initially for repair; however, if high levels of genomic U occur when UNG/SMUG1/MMR are not present then the cells would not initially die but over time would acquire a high amount of mutations, leading to either death or cancerous phenotypes. Though, a lot more work is needed to validate this hypothesis but visualising cell death in a UNG/SMUG1/MMR deficient cell with high levels of genomic U might reveal some answers.
