Mutator effects and mutation signatures of editing deaminases produced in bacteria and yeast.

Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenosine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This can not be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation.

UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae.

UVA (320-400 nm) radiation constitutes >90% of the environmentally relevant solar UV radiation, and it has been proposed to have a role in skin cancer and aging. Because of the popularity of UVA tanning beds and prolonged periods of sunbathing, the potential deleterious effect of UVA has emerged as a source of concern for public health. Although generally accepted, the impact of DNA damage on the cytotoxic, mutagenic, and carcinogenic effect of UVA radiation remains unclear. In the present study, we investigated the sensitivity of a panel of yeast mutants affected in the processing of DNA damage to the lethal and mutagenic effect of UVA radiation. The data show that none of the major DNA repair pathways, such as base excision repair, nucleotide excision repair, homologous recombination, and postreplication repair, efficiently protect yeast from the lethal action of UVA radiation. In contrast, the results show that the Ogg1 DNA glycosylase efficiently prevents UVA-induced mutagenesis, suggesting the formation of oxidized guanine residues. Furthermore, sequence analysis of UVA-induced canavanine-resistant mutations reveals a bias in favor of GC-->TA events when compared with spontaneous or H(2)O(2)-, UVC-, and gamma-ray- induced canavanine-resistant mutations in the WT strain. Taken together, our data point out a major role of oxidative DNA damage, mostly 7,8-dihydro-8-oxoguanine, in the genotoxicity of UVA radiation in the yeast Saccharomyces cerevisiae. Therefore, the capacity of skin cells to repair 7,8-dihydro-8-oxoguanine may be a key parameter in the mutagenic and carcinogenic effect of UVA radiation in humans.

Total synthesis of (-)-cylindrocyclophanes A and F exploiting the reversible nature of the olefin cross metathesis reaction.

Efficient total syntheses of the C(2)-symmetric (-)-cylindrocyclophanes A and F (1a and 1f) have been achieved. The initial strategy featured the use of a common advanced intermediate to assemble in stepwise fashion the required macrocycle of 1f, exploiting in turn a Myers reductive coupling followed by ring-closing metathesis. In a second-generation strategy, a remarkable cross olefin metathesis dimerization cascade was discovered and exploited to assemble the requisite [7,7]-paracyclophane macrocycles of both 1a and 1f from dienyl monomers. The successful syntheses also featured the effective use of the Danheiser annulation to construct substrates for both the Myers reductive coupling and the metathesis dimerizations strategies. Finally, the Kowalski two-step chain homologation of esters to siloxyalkynes proved superior over the original one-step protocol.

Efficient stereochemical relay en route to leucascandrolide A.

[structure: see text]. A complete relay of the initial stereochemical information is central to the efficient and highly stereocontrolled construction of the C1-C15 fragment of the marine macrolide leucascandrolide A. Cyclic silane 3, assembled via Pt-catalyzed hydrosilylation, was designed to serve as a temporary template for the installation of the C12 stereogenic center. The strategy features a highly convergent C10-C11 bond construction via 1,5-anti-selective aldol reaction and rapid assembly of the trisubstituted pyran subunit via Prins desymmetrization.

Hypersensitivity of Escherichia coli Delta(uvrB-bio) mutants to 6-hydroxylaminopurine and other base analogs is due to a defect in molybdenum cofactor biosynthesis.

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.

Racemic and asymmetric Diels-Alder reactions of 1-(2-oxazolidinon-3-yl)-3-siloxy-1,3-butadienes.

Achiral and chiral 1-(2-oxazolidinon-3-yl)-3-siloxy-1,3-butadienes were prepared from readily available starting materials. Although more stable than the parent 1-amino-3-siloxy dienes, the 1-(2-oxazolidinon-3-yl)-3-siloxy-1,3-butadienes are still very reactive in Diels-Alder reactions, somewhat more than 1,3-dialkoxy-1, 3-butadienes (e.g., Danishefsky's diene). The cycloadditions of the achiral and chiral dienes with several different dienophiles were examined. The reactions proceeded in good yield, with modest to high endo selectivity. The chiral dienes exhibited excellent facial selectivity in cycloadditions with alpha-substituted acroleins, maleic anhydride and N-phenylmaleimide. Upon reduction and hydrolysis of the cycloadducts, substituted cyclohexenones were obtained with ee's ranging from 22% to >98%.

Regiocontrolled synthesis of carbocycle-fused indoles via arylation of silyl enol ethers with o-nitrophenylphenyliodonium fluoride.

[formula: see text] A new, regiocontrolled synthesis of carbocycle-fused indoles has been developed. The two-step procedure involves first the regiospecific arylation of silyl enol ethers with o-nitrophenylphenyliodonium fluoride (1). Reduction of the nitro group on the aromatic ring with TiCl3 followed by spontaneous condensation of the aniline with the ketone then affords the indole products.

Multiple antimutagenesis mechanisms affect mutagenic activity and specificity of the base analog 6-N-hydroxylaminopurine in bacteria and yeast.

Base analog 6-N-hydroxylaminopurine is a potent mutagen in variety of prokaryotic and eukaryotic organisms. In the review, we discuss recent results of the studies of HAP mutagenic activity, genetic control and specificity in bacteria and yeast with the emphasis to the mechanisms protecting living cells from mutagenic and toxic effects of this base analog.

Overexpression of the yeast HAM1 gene prevents 6-N-hydroxylaminopurine mutagenesis in Escherichia coli.

The base analogue 6-N-hydroxylaminopurine (HAP) is a potent mutagen in a variety of prokaryotic and eukaryotic organisms. Mutations in the yeast ham1 gene render the cells hypersensitive to the mutagenic effect of HAP. We have found that this gene has homologues in a variety of organisms from bacteria to man. We have overexpressed yeast Ham1p in E. coli. We demonstrate that under conditions when this protein constitutes approximately 30% of cellular protein, the host strain is protected both from toxic and mutagenic effects of HAP. This result indicates that sole Ham1p activity might be sufficient for destruction of HAP or its metabolites in bacterial cells.

HAM1, the gene controlling 6-N-hydroxylaminopurine sensitivity and mutagenesis in the yeast Saccharomyces cerevisiae.

The ham1 mutant of yeast Saccharomyces cerevisiae is sensitive to the mutagenic and lethal effects of the base analog, 6-N-hydroxylaminopurine (HAP). We have isolated a clone from a centromere-plasmid-based genomic library complementing HAP sensitivity of the ham1 strain. After subcloning, a 3.4 kb functional fragment was sequenced. It contained three open reading frames (ORFs) corresponding to proteins 353, 197 and 184 amino acids long. LEU2+ disruptions of the promoter and N-terminal part of the gene coding 197 amino acids long protein led to moderate and strong sensitivity to HAP, respectively, and were allelic to the original ham1-1 mutation. Thus this ORF represents the HAM1 gene. The deduced amino acid sequence of HAM1 protein was not similar to any protein sequence of the SwissProt database. The HAM1 gene was localized on the right arm of chromosome X between cdc8 and cdc11. Spontaneous mutagenesis was not affected by the ham1::LEU2 disruption mutation.