Mouse 3-Methyladenine DNA Glycosylase Type II

CONTENTS:

Catalog # /Description /Size

4090-100-01 Aag protein 100 Units

3900-500-09 10X REC™ Buffer 9 1 ml

Mouse Aag is a 36 kDa constitutively expressed (1,000-2,000 copies/cell) protein. It acts on 3-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine, and a number of other substrates.

SOURCE:

Purified from E. coli containing a recombinant plasmid encoding the mouse Aag protein.

UNIT DEFINITION :

One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing hypoxanthine within an oligonucleotide duplex in one hour at 37°C.

SPECIFICITY:

Mouse Aag catalyzes the excision of the following forms of DNA damage: 3-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine, and 1,N6-ethenoadenine. It may alsofunction on the following forms of DNA damage: 7- and 3-ethylpurines,1-carboxyethyladenine, 7-carboxyethylguanine, O2-methylpyrimidines, 7(2-ethoxyethyl)-guanine,7(2-hydroxyethyl)guanine, 7(2-chloroethyl)guanine, 1,2-bis(7-guanyl)ethane, 3-ethylthioethylpurines, N2,3-ethenoguanine, N2,3-ethanoguanine, 5-hydroxymethyluracil, 5-formyluracil, 3,N4-ethenocytosine, 1,N2-ethenoguanine, 3,N2-etheno-guanine, and chloroacetaldehydecyclic adducts.

ASSAY CONDITIONS:

1X REC Buffer 9 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM EGTA,and 0.1 mM DTT), 4 pmole of labeled hypoxanthine oligonucleotide annealed to the compliment oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20μl areincubated for 1 hour at 37°C. To complete cleavage of an abasic site, fresh 1N NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24μlof 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue) are added, the samples are heated to 95°C for 10 minutes then fast cooled to 4°C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.

STORAGE BUFFER:

10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA, and 50% (v/v) glycerol.

STORAGE:

Store at -20°C in a manual defrost freezer.

REFERENCES:

1. Mattes, W.B., C.-S. Lee, J. Laval, and T.R. O'Conner. 1996. Excision of DNA adducts of nitrogen mustards by bacterial and mammalian 3-methyladenine-DNA glycosylases. Carcinogenesis 17:643-648.
2. Samson, L., B. Derfler, M. Boosalis, and K. Call. 1991. Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16. Proc. Natl. Acad. Sci. USA 88:9127-9131.