hUNG2 Is the Major Repair Enzyme for Removal of Uracil from U:A Matches, U:G Mismatches, and U in Single-stranded DNA, with hSMUG1 as a Broad Specificity Backup

Abstract

hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double-and singlestranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg 2؉, at which the activity of hUNG2 is 2-3 orders of magnitude higher than of hSMUG1. Moreover, Mg 2؉ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4 -ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup.Uracil in DNA can be introduced via two mechanisms, deamination of cytosine and misincorporation of dUMP during replication. Deamination of cytosine has been calculated from measured deamination rates to occur at a rate of 100 -500 per human cell/day (1, 2) to yield mutagenic U:G mispairs. Uracil may also appear as a consequence of misincorporation of dUMP instead of dTMP during replication, resulting in a U:A base pair. The latter is not miscoding, but may produce cytotoxic and mutagenic AP site intermediates during repair. In organisms containing 5-methylcytosine in their genomes, deamination of 5-methylcytosine furthermore leads to T:G mismatches. All living organisms express uracil-DNA glycosylases (UDGs) 1 that prevent cytotoxic and mutagenic effects of the above lesions. UDGs remove uracil (and sometimes other damaged bases or thymine) from the deoxyribose and thus initiate a multistep base excision repair (BER) pathway, eventually restoring the correct DNA sequence. After removal of uracil by an UDG and cleavage of the resulting abasic site by AP endonuclease (APE1/APE2), the BER pathway splits into two branches (reviewed in Ref.3). The presumed major track is the shortpatch pathway. It uses the 5Ј-deoxyribophosphodiesterase activity of DNA polymerase ␤ to cleave 3Ј of the abasic site, thus releasing deoxyribose-5-phosphate. Then pol ␤ inserts C or T, depending on the template base. Finally, DNA ligase III seals the nick, perhaps aided by the scaffold protein XRCC1. The alternative long-patch pathway largely uses replicatio...

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

“…Since the N-terminus of UNG2 was reported to be susceptible to proteolysis [39], UNG2 was produced in E. coli as an N-terminal his 6 -tag fusion protein. Following purification to apparent homogeneity (Fig.…”

Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil–DNA glycosylase impair learning in a mouse model of vascular cognitive impairment

“…Several enzymes are required to convert the damaged DNA [30]. One enzyme that plays a crucial role in this process is uracil-DNA glycosylase (UNG); it removes mismatched uracil from single and double stranded DNA [31]. Its inhibition in neuronal cultures results in neuronal death [32] and UNG knockout mice with elevated levels of homocysteine through dietary folate deficiency, show degeneration in the hippocampus as well as impairments in learning [33].…”

Repair of exocyclic DNA adducts: rings of complexity

“…(36,48,72,76) Overlap between glycosylases is also common as examplified by the in vitro identification of the eC-activity in three human DNA glycosylase, TDG, SMUG1 and MBD4. (52,(54)(55)(56) Although the biological function of these activities remains to be seen, such redundancy of repair is expected to be useful for backing up in vivo.…”

“…hTDG also excises eC paired with G with greater efficiency than T from T Á G, but less than U from U Á G. (52,54) Both proteins can remove eC opposite each of the four bases but with varying efficiencies, with eC Á G the preferred substrate. (52,54,57) Recently, Kavli et al (55) described excision of eC by the human single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), which is only found in higher eukaryotes. In another study, the human methyl-CpG binding domain protein (MBD4 or MED1) also shows a weak activity toward eC but only when the opposite base is G. (56) The biochemical details of these two activities have not been reported.…”