Specific interaction of wild-type and truncated mouse N-methylpurine- DNA glycosylase with ethenoadenine-containing DNA

Rabindra Roy, Tapan Biswas, Tapas Hazra, Gargi Roy, David T. Grabowski, Tadahide Izumi, Ganesan Srinivasan, Sankar Mitra

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, is responsible for the removal of a wide variety of alkylated base lesions in DNA, e.g., N-alkylpurines and cyclic ethenoadducts of adenine, guanine, and cytosine. These lesions, some of which are mutagenic and toxic, are generated endogenously or by genotoxic agents such as N-alkylnitrosamines and vinyl chloride. Wild-type mouse MPG, expressed from recombinant baculovirus, was purified to near homogeneity for studying its specific interaction with substrate, 1,N6-ethenoadenine- (̇A-) containing DNA. Electrophoretic mobility shift assays (EMSA) indicated that MPG formed a specific complex with a 50-mer ̇A containing duplex oligonucleotide. This complex was shown to be a transient reaction intermediate, because it could be formed only with the unreacted substrate and contained active enzyme molecules. DNA footprinting studies confirmed the specific binding of the protein to the εA-containing oligonucleotide; eight nucleotides on the εA-containing strand and 16-17 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion. A systemic deletion analysis of MPG was carried out in order to determine the minimally sized polypeptide capable of forming a stable substrate complex that is also suitable for characterization by NMR spectroscopy and X-ray crystallography. A truncated polypeptide (NΔ100CΔ18) lacking 100 and 18 amino acid residues from the amino and carboxyl termini, respectively, was found to be the minimal size that retained activity. The truncated and wild-type enzymes have similar kinetic properties. Moreover, both EMSA and DNase I footprinting studies indicated identical pattern of specific binding by the truncated and full- length polypeptides. Removal of five and nine additional residues from the amino- and carboxyl-termini of this polypeptide, respectively, resulted in a complete loss of activity. These results suggest that minimal structural change occurred as a result of truncation in the NΔ100CΔ18 mutant, which may thus be suitable for elucidating the structure and mechanism of MPG.

Original languageEnglish (US)
Pages (from-to)580-589
Number of pages10
JournalBiochemistry
Volume37
Issue number2
DOIs
StatePublished - Jan 13 1998

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DNA
Electrophoretic mobility
Peptides
Deoxyribonuclease I
Electrophoretic Mobility Shift Assay
Oligonucleotides
Assays
DNA Footprinting
Substrates
Nucleotides
DNA Repair Enzymes
Vinyl Chloride
Reaction intermediates
Baculoviridae
Poisons
Cytosine
X ray crystallography
X Ray Crystallography
Guanine
Adenine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specific interaction of wild-type and truncated mouse N-methylpurine- DNA glycosylase with ethenoadenine-containing DNA. / Roy, Rabindra; Biswas, Tapan; Hazra, Tapas; Roy, Gargi; Grabowski, David T.; Izumi, Tadahide; Srinivasan, Ganesan; Mitra, Sankar.

In: Biochemistry, Vol. 37, No. 2, 13.01.1998, p. 580-589.

Research output: Contribution to journalArticle

Roy, R, Biswas, T, Hazra, T, Roy, G, Grabowski, DT, Izumi, T, Srinivasan, G & Mitra, S 1998, 'Specific interaction of wild-type and truncated mouse N-methylpurine- DNA glycosylase with ethenoadenine-containing DNA', Biochemistry, vol. 37, no. 2, pp. 580-589. https://doi.org/10.1021/bi972313l
Roy, Rabindra ; Biswas, Tapan ; Hazra, Tapas ; Roy, Gargi ; Grabowski, David T. ; Izumi, Tadahide ; Srinivasan, Ganesan ; Mitra, Sankar. / Specific interaction of wild-type and truncated mouse N-methylpurine- DNA glycosylase with ethenoadenine-containing DNA. In: Biochemistry. 1998 ; Vol. 37, No. 2. pp. 580-589.
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AU - Izumi, Tadahide

AU - Srinivasan, Ganesan

AU - Mitra, Sankar

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