Schizosaccharomyces pombe Ofd2 Is a Nuclear 2-Oxoglutarate and Iron Dependent Dioxygenase Interacting with Histones

2-oxoglutarate (2OG) dependent dioxygenases are ubiquitous iron containing enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. They participate in a wide range of biological processes including collagen biosynthesis, fatty acid metabolism, hypoxic sensing and demethylation of nucleic acids and histones. Although substantial progress has been made in elucidating their function, the role of many 2OG dioxygenases remains enigmatic. Here we have studied the 2OG and iron (Fe(II)) dependent dioxygenase Ofd2 in Schizosaccharomyces pombe, a member of the AlkB subfamily of dioxygenases. We show that decarboxylation of 2OG by recombinant Ofd2 is dependent on Fe(II) and a histidine residue predicted to be involved in Fe(II) coordination. The decarboxylase activity of Ofd2 is stimulated by histones, and H2A has the strongest effect. Ofd2 interacts with all four core histones, however, only very weakly with H4. Our results define a new subclass of AlkB proteins interacting with histones, which also might comprise some of the human AlkB homologs with unknown function

Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

Background: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. Methodology and Findings: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB2 mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. Conclusions: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis

Mice Lacking Alkbh1 Display Sex-Ratio Distortion and Unilateral Eye Defects

Escherichia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases.In the present study we show non-Mendelian inheritance of the Alkbh1 targeted allele in mice. Both Alkbh1(-/-) and heterozygous Alkbh1(+/-) offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one female born for every three to four males. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes, apoptotic spermatids were revealed in 5-10% of the tubules in Alkbh1(-/-) adults. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant phenotypes observed, particularly recurrent unilateral eye defects and craniofacial malformations.Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio distribution and embryonic development in mice