Properties of a Recombinant Human Uracil-DNA Glycosylase from the UNG Gene and Evidence that UNG Encodes the Major Uracil-DNA Glycosylase

Abstract

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

DNA glycosylases in the base excision repair of DNA

“…The rate differs as much as 20-fold between the ' best ' (A\T\UAA) and ' worst ' (G\CUG\C\T) sequences. Usually, but not always [34], the rate of removal is greater from U\G mispairs than from U :A pairs [35][36][37]. A low removal rate tends to be correlated with the occurrence of ' hot spots ' for mutation [36], similar to the correlation between slow spots for the repair of cyclobutyl dimers and hot spots for mutations [38,39].…”

“…Ugi forms an essentially irreversible complex with UDG [68,69], and the specificity of the interaction [70,71] indicates that only structurally related UDGs will bind to Ugi. Furthermore, antibodies raised against homogeneous human UDG detected both mitochondrial and nuclear forms, indicating that these forms are closely related [37,64,72].…”

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

“…The core catalytic domain of UNG (UNGΔ84), which lacks the N-terminal 84 amino acids of UNG2 [5], was overproduced in E. coli and purified as described by Chen et al [35]. PCNA LC59 was overproduced in E. coli BL21 (DE3) and purified as described previously for wild-type PCNA, except that the LC59 double mutant flowed through, rather than binding to, the phenyl-Sepharose column [32].…”

“…required for subcellular sorting, while the core catalytic domain (UNGΔ84) is common to the two forms [3,4]. All forms of UNG examined remove uracil opposite A or G in doublestranded DNA as well as from single-stranded DNA [3,5]. At least four different human nuclear DNA glycosylases, UNG2, single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), thymine-DNA glycosylase (TDG) and methyl-CpG binding domain protein 4 (MBD4, also known as MED1) have been reported to removal uracil from U·G mispairs [6][7][8], and it has not yet been determined whether the different uracil-DNA glycosylases have distinct or overlapping physiological functions.…”

Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

“…UNG proteins are relatively small monomeric proteins that usually do not require cofactors or ions for their activity. They are also highly specific against uracil in both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), with a preference for ssDNA (64,75).…”