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.

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.

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.

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.

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.