Nuclear and mitochondrial uracil-DNA glycosylases are generated by alternative splicing and transcription from different positions in the UNG gene

Abstract

A distinct nuclear form of human uracil-DNA glycosylase [UNG2, open reading frame (ORF) 313 amino acid residues] from the UNG gene has been identified. UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino acids of the N-terminal sequence, which is not necessary for catalytic activity. The rest of the sequence and the catalytic domain, altogether 269 amino acids, are identical. The alternative N-terminal sequence in UNG2 arises by splicing of a previously unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1 sequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed in mouse which has an identical organization of the homologous gene. Constructs that express fusion products of UNG1 or UNG2 and green fluorescent protein (EGFP) were used to study the significance of the N-terminal sequences in UNG1 and UNG2 for subcellular targeting. After transient transfection of HeLa cells, the pUNG1-EGFP-N1 product colocalizes with mitochondria, whereas the pUNG2-EGFP-N1 product is targeted exclusively to nuclei.

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

“…Burgers and Yoder [47] reported that PCNA could stimulate synthesis by DNA polymerase δ on linear templates by slipping onto the end of the DNA in an ATP-independent manner, although a large molar excess was required for maximal stimulation. Since large amounts (810 pmol) of PCNA were required in order to detect stimulation of UNG2 activity, was passive end-loading of PCNA onto the calf thymus [uracil- 3 810 pmol), and the amount of excised 3 H uracil was quantified as described under Section 2; the results are shown in Fig. 7A.…”

“…In human cells, the UNG gene encodes both the nuclear form (UNG2) and the mitochondrial pre-form (UNGl) of uracil-DNA glycosylase using different promoters and alternative splicing [3]. UNGl and UNG2 have unique N-terminal domains ☆ This work was supported by National Institute of General Medical Sciences Grant GM66245.…”

“…UNGl and UNG2 have unique N-terminal domains ☆ This work was supported by National Institute of General Medical Sciences Grant GM66245. 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].…”

“…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.…”

Mitochondria, oxidative DNA damage, and aging

“…This activity, which excises uracil from DNA, was later named UDG1 when it was purified from human cells by affinity chromatography. It is a 30kd protein, encoded by the same gene which encodes the 36kd nuclear form of the protein (157,158).…”