The Domains of Mammalian Base Excision Repair Enzyme N-Methylpurine-DNA Glycosylase: INTERACTION, CONFORMATIONAL CHANGE, AND ROLE IN DNA BINDING AND DAMAGE RECOGNITION

Rabindra Roy,Amalendra Kumar,J Ching Lee,Sankar Mitra

doi:10.1074/jbc.271.39.23690

Rabindra Roy, Amalendra Kumar + Show 2 more

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https://doi.org/10.1074/jbc.271.39.23690

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Abstract

Repair of a variety of alkylated base adducts in DNA is initiated by their removal by N-methylpurine-DNA glycosylase. The 31-kDa mouse N-methylpurine-DNA glycosylase, derived by deletion of 48 amino acid residues from the 333-residue wild type protein without loss of activity, was analyzed for the presence of protease-resistant domains with specific roles in substrate binding and catalysis. Increasing proteolysis with trypsin generated first a 29-kDa polypeptide by removal of 42 amino-terminal residues, followed by production of 8-, 6-, and 13-kDa fragments with defined, nonoverlapping boundaries. The 8- and 13-kDa domains include the amino and carboxyl termini, respectively. Based on DNA-affinity chromatography and the protease protection assay, it appears that the 6- and 13-kDa domains are necessary for nontarget DNA binding and that the 8-kDa domain, in cooperation with the other two domains, participates in recognition of damaged bases. Furthermore, chemical cross-linking studies indicated that, in the presence of substrate DNA, the 8- and 6-kDa domains undergo conformational changes reflected by both protection from proteolysis and reduced availability of cysteine residues for the thiol-exchange reaction.

Highlights

Repair of a variety of alkylated base adducts in DNA is initiated by their removal by N-methylpurine-DNA glycosylase
How do these domains interact during substrate binding and catalysis? In this study controlled proteolysis was employed to identify the domains of mouse MPG in solution and to characterize their possible functions
To acquire a full range of near- and far-ultraviolet (UV) circular dichroism (CD) spectra, fused quartz cuvettes with path lengths of 0.01 and 0.1 cm were used for protein and DNA solutions with concentrations around 10Ϫ5 M

Summary

Introduction

Repair of a variety of alkylated base adducts in DNA is initiated by their removal by N-methylpurine-DNA glycosylase. The 31-kDa mouse N-methylpurine-DNA glycosylase, derived by deletion of 48 amino acid residues from the 333-residue wild type protein without loss of activity, was analyzed for the presence of protease-resistant domains with specific roles in substrate binding and catalysis. The mouse MPG (and the aminoterminal half of the mouse MPG in a mouse/human hybrid protein) showed repair of 7-methylguanine and 3-methylguanine at a higher rate than did human MPG or the aminoterminal half of human MPG in human/mouse hybrid protein (when the levels of both proteins were adjusted to release 3-methyladenine at comparable rate) This result indicates that the amino-terminal half of MPG is more critical for the recognition of 3-methylguanine and 7-methylguanine [31]. How do these domains interact during substrate binding and catalysis? In this study controlled proteolysis was employed to identify the domains of mouse MPG in solution and to characterize their possible functions

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