Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae.

The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway. The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S. cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H. J. You, R. L. Swanson, and P. W. Doetsch, Biochemistry 37:6033-6040, 1998). The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast. We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione. The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents. The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway. In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively. These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S. cerevisiae. In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.

Yeast base excision repair: interconnections and networks.

The removal of oxidative base damage from the genome of Saccharomyces cerevisiae is thought to occur primarily via the base excision repair (BER) pathway in a process initiated by several DNA N-glycosylase/AP lyases. We have found that yeast strains containing simultaneous multiple disruptions of BER genes are not hypersensitive to killing by oxidizing agents, but exhibit a spontaneous hyperrecombinogenic (hyper-rec) and mutator phenotype. The hyper-rec and mutator phenotypes are further enhanced by elimination of the nucleotide excision repair (NER) pathway. Furthermore, elimination of either the lesion bypass (REV3-dependent) or recombination (RAD52-dependent) pathway results in a further, specific enhancement of the hyper-rec or mutator phenotypes, respectively. Sensitivity (cell killing) to oxidizing agents is not observed unless multiple pathways are eliminated simultaneously. These data suggest that the BER, NER, recombination, and lesion bypass pathways have overlapping specificities in the removal of, or tolerance to, exogenous or spontaneous oxidative DNA damage in S. cerevisiae. Our results also suggest a physiological role for the AP lyase activity of certain BER N-glycosylases in vivo.

Heparin-induced thrombocytopenia: IgG binding to PF4-heparin complexes in the fluid phase and cross-reactivity with low molecular weight heparin and heparinoid.

Early diagnosis of heparin-induced thrombocytopenia (HIT) is essential to reduce morbidity and mortality. We report an enzyme immunoassay which detects the binding of HIT IgG to PF4-heparin in the fluid phase. Our fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. We were able to detect anti-PF4-heparin IgG in 26/28 (93%) HIT patients. We investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 23/26 (88%) of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.

Larval desperation and histamine: how simple responses can lead to complex changes in larval behaviour.

Some marine invertebrate larvae expand the range of settlement cues to which they will respond as they age. How do relatively simple larvae achieve such complex changes in behaviour? Larvae of the Australian sea urchin Holopneustes purpurascens settle and metamorphose specifically in response to a settlement cue, dissolved histamine, produced by the host alga Delisea pulchra. Older H. purpurascens larvae appear to accept a wider range of host algae, which contain far less histamine than D. pulchra, than newly competent larvae. We tested the hypothesis that older H. purpurascens larvae accept a greater range of host algae by metamorphosing in response to lower concentrations of histamine. We compared the response of newly competent and older larvae to a range of histamine concentrations in settlement assays. Larval age strongly affected the minimum concentration of histamine that induced metamorphosis in H. purpurascens, with older larvae responding to lower concentrations of histamine than newly competent larvae. Older larvae were more sensitive to lower concentrations of histamine yet still maintained a stringent requirement for exposure to histamine in order to metamorphose. In addition, older larvae metamorphosed after shorter exposure periods to histamine than did younger larvae. By using histamine concentration as a proxy for specific habitat cues, H. purpurascens larvae appear to expand their range of settlement preferences with age by simply changing their sensitivity to a single settlement cue. Overall, our results show that marine invertebrate larvae can exhibit surprisingly complex changes in behaviour via simple changes in their response to a single cue.

Dual-lock total hip arthroplasty. A preliminary experience.

The long-term experience with total hip arthroplasty (THA) has shown an increasing failure rate associated with aseptic loosening of components. A Dual-Lock system incorporates features designed to reduce the incidence of loosening. The Dual-Lock THA has a low-profile acetabular component that permits preservation of subchondral bone. The design features a large, collarless, straight-stem femoral component with an interlocking metal-to-bone press fit as the primary form of fixation. In a series of 210 THAs in 180 patients, with follow-up examinations over a period of one to four years, the results were 92% good or excellent. A problem with the press fit is femoral cracking. Subsidence, early loosening, and calcar changes have not been found to a significant degree. Preliminary results with this new design have been encouraging and support the concept of the press fit.

Saccharomyces cerevisiae possesses two functional homologues of Escherichia coli endonuclease III.

We previously identified two distinct genes of Saccharomyces cerevisiae redoxyendonuclease (SCR1 and SCR2) which possess a high degree of sequence similarity to Escherichia coli endonuclease III [Augeri, L., Lee, Y. M., Barton, A. B., and Doetsch, P. W. (1997) Biochemistry 36, 721-729]. The proteins encoded by SCR1 and SCR2 were overexpressed in E. coli and purified to apparent homogeneity. Both proteins recognized and cleaved DNA substrates containing dihydrouracil, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (FaPy-7-MeGua), and abasic sites but not DNA substrates containing uracil or 8-oxoguanine. Purified Scr2, but not Scr1, possesses spectral properties which indicate the presence of an iron-sulfur center. Kinetic parameters for Scr1 and Scr2 were determined by using an oligonucleotide containing a single dihydrouracil. Analysis of the deduced amino acid sequences of Scr1 and Scr2 suggests that Scr2 bears an iron-sulfur motif, while Scr1 does not have this motif. However, Scr1 has a long, positively charged N-terminus that could be a mitochondrial transit sequence. Targeted gene disruption of SCR1 and SCR2 produced a double mutant that had no detectable enzymatic activity against the dihydrouracil-containing substrate. Northern blot analysis showed that SCR1 was induced by menadione, but SCR2 was not. These results indicate that although Scr1 and Scr2 are both functional homologues of E. coli endonuclease III, they differ from each other with respect to their amino acid sequences and inducibility by DNA damaging agents, suggesting that their precise biological roles may be different.

Very long-chain fatty acids change the ethanol tolerance of Drosophila melanogaster larvae.

This research tested the hypothesis that long-chain saturated fatty acids increase the order of cell membranes of an organism and minimize the detrimental fluidizing effects of ethanol. Unsaturated fatty acids increase membrane fluidity and are unlikely to increase the ethanol tolerance of the organism. Both a fatty acid-free medium and media supplemented with very long-chain fatty acids (20 or more carbons) were fed to wild-type larvae of Drosophila melanogaster; larvae were then transferred to media with or without ethanol to test for effects of the fatty acids on ethanol tolerance. Ethanol decreased the percent of larvae to pupate, and lengthened larval development time. However, the percentage of pupae to reach the adult stage and the weight of adult males increased when the larvae were fed ethanol. The very long-chain, unsaturated fatty acids, arachidonic acid [20:4(n-6)] and docosatetraenoic acid [22:4(n-6)], were associated with increased larval mortality when administered in a medium supplemented with ethanol. Arachidic acid (20:0) increased the percentage of larvae to pupate under ethanol stress, decreased the development time and increased the adult weight in the presence and absence of ethanol. Behenic acid (22:0) was not effectively incorporated into phospholipids and had little effect on growth traits. Thus, the experimental results were consistent with the hypothesis.

Saccharomyces cerevisiae Ntg1p and Ntg2p: broad specificity N-glycosylases for the repair of oxidative DNA damage in the nucleus and mitochondria.

Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg2p) of the Escherichia coli endonuclease III protein, a DNA base excision repair N-glycosylase with a broad substrate specificity directed primarily against oxidatively damaged pyrimidines. The substrate specificities of Ntg1p and Ntg2p are similar but not identical, and differences in their amino acid sequences as well as inducibility by DNA damaging agents suggest that the two proteins may have different biological roles and subcellular locations. Experiments performed on oligonucleotides containing a variety of oxidative base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substrate for Ntg2p. Vectors encoding Ntg1p-green fluorescent protein (GFP) and Ntg2p-GFP fusions under the control of their respective endogenous promoters were utilized to observe the subcellular targeting of Ntg1p and Ntg2p in S. cerevisiae. Fluorescence microscopy of pNTG1-GFP and pNTG2-GFP transformants revealed that Ntg1p localizes primarily to the mitochondria with some nuclear localization, whereas Ntg2p localizes exclusively to the nucleus. In addition, the subcellular location of Ntg1p and Ntg2p confers differential sensitivities to the alkylating agent MMS. These results expand the known substrate specificities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediate different roles in the repair of DNA base damage in the nucleus and mitochondria of yeast.