Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3, and H4; HCS knockdown disturbs gene regulation, and decreases stress resistance and life span in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast-two-hybrid studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by rHCS in vitro, based on tracing histone-bound biotin with [3H]biotin, streptavidin, and anti-biotin antibody. Biotinylation sitespecific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks, or chromatin proteins.

Arylhydrazides, arylhydrazines, and N-alkyl-N-arylnitrosamines are metabolized to arenediazonium ions which yield C8-arylpurine adducts in calf thymus and cellular DNA. The mechanism of adduct formation has not been fully elucidated. C8-Arylguanine adducts likely form from direct aryl radical (Ar*) addition to the C8 position of guanine. However, the amounts of C8-aryladenine adducts measured here are inconsistent with direct radical attack at the C8 position of adenine. An intermediate product, an aryltriazene, is likely formed which then decomposes to the C8-aryladenine adduct. We have demonstrated that N1-aryl-N3-purinyltriazene adducts are formed from a variety of para-substituted arenediazonium ions with adenine. Decomposition of the N1-aryl-N3-purinyltriazene, at high pH and elevated temperatures, has been shown to give C8-aryladenine derivatives, and a free radical mechanism for this process has been proposed. Here we show that this process can occur under physiological conditions and that the C8-aryladenine adduct can be quantitated by HPLC. ESR studies, in which DMPO was used as a spin trap, have been used to demonstrate the intermediacy of aryl radicals during the decomposition of the N1-aryl-N3-purinyltriazenes and to demonstrate that this process also occurs in calf thymus (ct) DNA treated with arenediazonium ions. These results suggest the involvement of an aryl radical in the formation of the observed DNA adducts. Finally, we have found that the treatment of ct DNA with arenediazonium ions produces a significant amount of depurination. Both the formation of C8-arylguanine and C8-aryladenine adducts and the generation of apurinic sites may contribute to the genotoxicity of arylhydrazides, arylhydrazines, N-alkyl-N-arylnitrosamines, and arenediazonium ions.

Sanitization of the cellular nucleotide pools from mutagenic base analogs is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic and toxic incorporation of purine base analogs (i.e. ITP, dITP, XTP, dXTP or 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele 94C->A encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analog drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by western blot is severely diminished in a human fibroblast cell line with the 94C->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability and availability.