The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH 2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 ؋ 10 4 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the K m was 47 nM, and k cat was ϳ0.6/min, independent of whether DHU paired with G or A. The enzyme carries out ␤-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptideoligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.Reactive oxygen species are generated as by-products of oxidative phosphorylation or by ionizing radiation and induce extensive base damage that is mainly repaired via the base excision repair (BER) 1 pathway. This repair is initiated by removal of the damaged base, catalyzed by a DNA glycosylase (1-3). There are two classes of DNA glycosylases with distinct substrate specificities: the monofunctional simple glycosylase and the glycosylase with associated AP lyase activity. All oxidized base lesions are removed from DNA by DNA glycosylase/AP lyases, which not only catalyze removal of the base lesion but also cause strand cleavage via ␤-elimination. The Escherichia coli endonuclease III (Nth) recognizes a wide range of oxidized pyrimidine derivatives, including ring-saturated and ring-fragmented derivatives such as thymine glycol (Tg), 5-hydroxycytosine, 5,6-dihydrouracil (DHU), and urea (1, 3-6). This enzyme has been well conserved from E. coli to the humans (7,8). On the other hand, oxidized purine lesions are also repaired by DNA glycosylase/AP lyases, i.e. Mut M (Fpg) of E. coli or OGG of eukaryotes (yeast and mammals), which do not share extensive sequence similarity. Furthermore, unlike these e...

Background: Akabane virus is a member of the genus Orthobunyavirus in the family Bunyaviridae. It is transmitted by hematophagous arthropod vectors such as Culicoides biting midges and is widely distributed in temperate to tropical regions of the world. The virus is well known as a teratogenic pathogen which causes abortions, stillbirths, premature births and congenital abnormalities with arthrogryposishydranencephaly syndrome in cattle, sheep and goats. On the other hand, it is reported that the virus rarely induces encephalomyelitis in cattle by postnatal infection. A first large-scale epidemic of Akabane viral encephalomyelitis in cattle occurred in the southern part of Japan from summer to autumn in 2006. The aim of this study is to define the epidemiological, pathological and virological properties of the disease.

The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) plays a central role in the DNA base excision repair pathway (BER) in two distinct ways. As an AP endonuclease, it initiates repair of AP sites in DNA produced either spontaneously or after removal of uracil and alkylated bases in DNA by monofunctional DNA glycosylases. Alternatively, by acting as a 3'-phosphoesterase, it initiates repair of DNA strand breaks with 3'-blocking damage, which are produced either directly by reactive oxygen species (ROS) or indirectly through the AP lyase reaction of damage-specific DNA glycosylases. The endonuclease activity of APE1, however, is much more efficient than its DNA 3'-phosphoesterase activity. Using whole extracts from human HeLa and lymphoblastoid TK6 cells, we have investigated whether these two activities differentially affect BER efficiency. The repair of ROS-induced DNA strand breaks was significantly stimulated by supplementing the reaction with purified APE1. This enhancement was linearly dependent on the amount of APE1 added, while addition of other BER enzymes, such as DNA ligase I and FEN1, had no effect. Moreover, depletion of endogenous APE1 from the extract significantly reduced the repair activity, suggesting that APE1 is essential for repairing such DNA damage and is limiting in extracts of human cells. In contrast, when uracil-containing DNA was used as the substrate, the efficiency of repair was not affected by exogenous APE1, presumably because the AP endonuclease activity was not limiting. These results indicate that the cellular level of APE1 may differentially affect repair efficiency for DNA strand breaks but not for uracil and AP sites in DNA.

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