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Identifying Genetic Determinants of ALKBH2 Evolvability

Abstract

ALKBH2 is an Fe(II), alpha-ketoglutarate dependent dioxygenase that repairs methyl and etheno lesions. Recently, ALKBH2 has been described as a tumor suppressor, and somatic ALKBH2 mutations have been reported in tumors. To understand selective pressures driving ALKBH2 evolution, we evolved an ALKBH2 mutant library under MNNG selection, which generates methyl adducts that are not known ALKBH2 substrates. Mutants from our selection overlap with tumor mutations. We used E. coli complementation systems to study how these mutations alter protection against both known methyl and etheno substrates, and against non-canonical cytotoxic lesions. We find that: 1) ALKBH2 mutant activity against known substrates is decreased but not abolished 2) Activity against methyl adducts can be differentially modulated relative to etheno adducts. 3) Some of our MNNG-selected mutations exhibit protection to additional cytotoxic lesions. These findings open the door for genetic modulation of ALKBH2 substrate specificity, which has implications for alkylating agent therapy.

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