The role of amino-terminal domain of RAD51C in promoting ALKBH3 DNA repair activity

Rukmini, Sarma and Roy, Anindya (2019) The role of amino-terminal domain of RAD51C in promoting ALKBH3 DNA repair activity. Masters thesis, Indian institute of technology Hyderabad.

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Abstract

DNA alkylating agents are found in industrial chemicals, environmental contaminants, and chemotherapeutic drugs. These chemicals alkylate nucleophilic sites in DNA, mostly nitrogen and oxygen. If not repaired, DNA alkylation leads to mutation and cytotoxicity. Human DNA repair protein ALKBH3 catalyses the direct reversal of N-alkyl-adducts from single-stranded DNA by an oxidative demethylation reaction requiring α-ketoglutarate and Fe2+ as cofactors. Recent interaction studies have revealed that there is a direct protein-protein interaction between ALKBH3 and homologous recombination protein RAD51C. At the molecular level, ALKBH3-RAD51C interaction stimulates the repair activity of ALKBH3. To investigate whether the amino terminal domain of RAD51C is involved in the interaction, we generated a deletion construct of RAD51C lacking the amino-terminal domain (RAD51C ΔNTD) and tested its ability to stimulate ALKBH3 activity. The deletion construct was PCR amplified, cloned in a suitable vector and the recombinant RAD51C ΔNTD protein was purified. Subsequently, demethylation assay was carried out with ALKBH3 in combination with RAD51C and RAD51C ΔNTD. The results included here show that ALBKH3-RAD51C interaction stimulates the ALKBH3 activity, while the ALKBH3-RAD51C ΔNTD interaction had no effect on the repair activity of ALKBH3, suggesting that ALBKH3-RAD51C interaction indeed takes place through the N-terminal domain of RAD51C.

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