Uracil‐DNA glycosylase is the DNA repair enzyme responsible for the removal of uracil from DNA, and it is present in all organisms investigated. Here we report on the cloning and sequencing of a cDNA encoding the human uracil‐DNA glycosylase. The sequences of uracil‐DNA glycosylases from yeast, Escherichia coli, herpes simplex virus type 1 and 2, and homologous genes from varicella‐zoster and Epstein‐Barr viruses are known. It is shown in this report that the predicted amino acid sequence of the human uracil‐DNA glycosylase shows a striking similarity to the other uracil‐DNA glycosylases, ranging from 40.3 to 55.7% identical residues. The proteins of human and bacterial origin were unexpectedly found to be most closely related, 73.3% similarity when conservative amino acid substitutions were included. The similarity between the different uracil‐DNA glycosylase genes is confined to several discrete boxes. These findings strongly indicate that uracil‐DNA glycosylases from phylogenetically distant species are highly conserved.

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.

Background: The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants.

Uracil-DNA glycosylase (UDG) is the first enzyme in the excision repair pathway for removal of uracil in DNA. In vitro transcription/translation of a cloned human cDNA encoding UDG resulted in easily measurable UDG activity. The apparent size of the primary translation product was 34 kD. Two lines of evidence indicated that this cDNA encodes the major nuclear UDG. First, in vitro translation of human fibroblast mRNA isolated from S-phase cells resulted in measurable UDG activity and this UDG translation was specifically inhibited 90% by an anti-sense UDG mRNA transcript. Secondly, cell cycle analysis revealed an 8-12 fold increase in transcript level late in the G1-phase preceding a 2-3 fold increase in total UDG activity in the S-phase. UDG degradation was found to be very slow (T1/2 approximately 30h), therefore, the rate of UDG synthesis could be derived from the rate of UDG accumulation, and was found to correlate temporarily and quantitatively with the transcript level. Inhibitor studies showed that RNA and protein synthesis was required for induction of UDG. However, specific inhibition of DNA replication with aphidicolin indicated that entrance of fibroblasts into the S-phase was not required for UDG accumulation.