An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.     

Intrarectal transmission of simian immunodeficiency virus in rhesus macaques: selective amplification and host responses to transient or persistent viremia.

Intrarectal simian immunodeficiency virus (SIV) infection in rhesus macaques is a model for sexual transmission of primate retroviruses. Phylogenetic studies on envelope gene sequences that were present in blood following intrarectal SIV inoculation provided evidence for selective amplification of a subset of viruses present in the inoculum and defined one amino acid sequence uniquely associated with intrarectal infection. Both persistent and transient viremia states were observed after intrarectal infection. Immune responses in persistently infected animals accounted for slower rates of disease progression despite the presence of highly pathogenic viruses that were documented by transfusion studies. Transient viremia elicited protective immunity against subsequent intrarectal virus challenge but did not protect against intravenous virus challenge. Transient viremia usually but not always led to self-limiting infection. In one animal, we documented a relapse to active viremia long after the initial transient viremia. SIV transmission across mucosal barriers affects pathogenesis in the short term by limiting the types of viruses established in the host and in the longer term by establishing host responses that slow disease progression despite the presence of highly pathogenic viruses in blood.     

Physiological Studies of the Rumen Protozoan Ophryoscolex caudatus Eberlein

The rumen ciliate Ophryoscolex caudatus fermented starch with the production of acetic, butyric, and lactic acids plus CO₂ and H₂. Cellulose was not significantly metabolized although pectin was rapidly attacked in the Warburg apparatus. The protein sources, cottonseed, soybean, and linseed oil meals, and the amino acids, dl-alanine, dl-valine, and dl-leucine, were utilized by the protozoan, whereas ammonia was demonstrated as an end product of nitrogenous metabolism. Methods for the separation of O. caudatus from mixed rumen contents are described.     

A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities.

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.     

Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage.

The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.     

Nuclease SP: a novel enzyme from spinach that incises damaged duplex DNA preferentially at sites of adenine.

A novel endonuclease has been isolated from extracts of spinach leaves (Spinacia oleracea). The enzyme has been purified by a series of column chromatography steps and has a molecular size of approximately 43,000 daltons. The spinach endonuclease cleaved double stranded DNA damaged by ultraviolet light or cis-diamminedichloroplatinum (II) primarily at sites of adenine when end-labelled DNA fragments of defined sequence were employed as substrates. The nature of the structural distortion contained in damaged, duplex DNA appears to be an important determinant for endonuclease cleavage. DNA helical distortions produced by UV light-induced (6-4) pyrimidine-pyrimidone photoproducts, but not cyclobutane pyrimidine dimers are recognized by the enzyme. The DNA cleavage products generated by the enzyme contain 3'-hydroxyl and 5'-phosphoryl termini. Single stranded DNA and RNA are hydrolyzed by the spinach endonuclease. This enzyme, which we call nuclease SP, is similar in several respects to other single-strand-specific nucleases such as N. crassa and mung bean nucleases and may function in DNA repair and/or recombination events in spinach cells. Nuclease SP should be a useful tool for the analysis of (6-4) photoproducts occurring in duplex DNA.