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

Mielecki, Damian and Zugaj, Dorota Łucja and Muszewska, Anna and Piwowarski, Jan and Chojnacka, Aleksandra and Mielecki, Marcin and Nieminuszczy, Jadwiga and Grynberg, Marcin and Grzesiuk, Elzbieta (2012) Novel AlkB Dioxygenases – Alternative Models for In Silico and In Vivo Studies. PloS one, 7 (1). e30588. ISSN 1932-6203

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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.

Item Type:Article
Subjects:Q Science > Q Science (General)
Q Science > QK Botany
Q Science > QP Physiology
Q Science > QR Microbiology
Divisions:Department of Genetics
Department of Molecular Biology
Department of Protein Biosynthesis
ID Code:298
Deposited By: Prof. Elżbieta Grzesiuk
Deposited On:24 Sep 2012 21:15
Last Modified:17 Oct 2014 14:08

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