Germ-line variants of human N-methylpurine DNA glycosylase show impaired DNA repair activity and facilitate 1,N6 ethenoadenine induced mutations [Genomics and Proteomics]

December 23rd, 2014 by Adhikari, S., Chetram, M. A., Woodrick, J., Mitra, P. S., Manthena, P. V., Khatkar, P., Dakshanamurthy, S., Dixon, M., Karmahapatra, S. K., Nuthalapati, N. K., Gupta, S., Narasimhan, G., Mazumder, R., Loffredo, C. A., Uren, A., Roy, R.

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1, N6-etheno adenine, hypoxanthine and alkylation adducts in DNA. Genetic studies discovered at least 8 validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations, which result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine (R) residues, R141 and R120, are important for hMPG s activity, as the germ-line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared to the wt hMPG. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG towards 1, N6-ethenoadenine containing oligonucleotide (εA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple-turnover (MTO) condition. SPR spectroscopy further showed that both variants had significantly decreased non-specific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between εA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence
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