Question

Q) You want to express human uracil in bacteria in glycosylase in bacteria. Besides the coding DNA for the glycosylase gene, what must be present in plasmid?

Answer 1

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.

Uracil is a common base lesion in DNA and is introduced into the genome by deamination of cytosine and misincorporation of dUMP instead of dTMP during replication. Spontaneous deamination of cytosine has been estimated to occur at a rate of 60–500 events per day in human cells (1–3). In addition, recent research has revealed that enzymatic deamination of cytosine at the Ig loci by activation-induced cytosine deaminase (AID) initiates the antigen-dependent antibody diversification processes (4). Uracil generated by deamination of cytosine is 100% miscoding, and result in C:G to T:A transition mutations if not repaired prior to replication. Misincorporated uracil is not directly miscoding, but it appears to be a critical source of spontaneously generated AP-sites (apurinic/apyrimidinic-sites) in the genome (5).

Uracil and some uracil analogs generated by oxidation of cytosine are excised from the genome by uracil-DNA glycosylases (UDGs). Mammalian cell nuclei contain at least four UDGs; UNG2, SMUG1, TDG and MBD4. Current evidence suggests that UNG2 (uracil-N-glycosylase 2) and SMUG1 (single-strand-selective monofunctional UDG) are the major enzymes responsible for repair of spontaneously deaminated cytosine (6–8), while post-replicative excision of misincorporated dUMP (U:A) and excision of AID-generated uracil (U:G) are performed mainly by UNG2 alone (9–11). Consistent with the role of UNG2 in replication associated repair, UNG2 binds PCNA and RPA, is localized to replication foci, and is cell cycle regulated with the highest levels in S-phase (9,12–15). Conversely, SMUG1, is not cell cycle regulated and is evenly distributed in the nucleoplasm (7). SMUG1 excises uracil from DNA with a much lower efficiency than UNG2, but has broader substrate specificity. Only SMUG1 excises thymine with an oxidized methyl group (7,16). UNG and SMUG1 belong to a superfamily that has apparently evolved from the same ancestral gene (17). Comparison of crystal structures of human UNG and Xenopus SMUG1 has revealed that these enzymes share a common fold and that the SMUG1 active site is a mosaic of features from UNG and MUG/TDG enzyme families (18).