PMID: 9445381Mar 7, 1998Paper

Probing of conformational changes in human O6-alkylguanine-DNA alkyl transferase protein in its alkylated and DNA-bound states by limited proteolysis

The Biochemical Journal
S KanugulaA E Pegg

Abstract

Human O6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair protein that protects cells from alkylation damage by transferring an alkyl group from the O6-position of guanine to a cysteine residue in the active site (-PCHR-) of the protein. The structure of the hAGT protein (23 kDa) has been probed by limited proteolysis with trypsin and Glu-C endoproteases and analysis of the polypeptide fragments by SDS/PAGE. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. Initial cleavage by trypsin occurred at residue Lys-193 to give a 21 kDa polypeptide fragment, and this polypeptide underwent further cleavage at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA (dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-128 position was less available for digestion in the presence of single-stranded DNA (ssDNA), suggesting that the hAGT protein has a different conformation when bound to ssDNA compared with dsDNA. When protease digestion was carried out on wild-type protein, preincubated with ...Continue Reading

Citations

Jul 11, 2006·Magnetic Resonance in Chemistry : MRC·Anne RobertsDavid E Wemmer
Oct 27, 2015·Extremophiles : Life Under Extreme Conditions·Antonella VettoneGiuseppe Perugino
Dec 24, 2002·The Journal of Biological Chemistry·Joseph J RasimasMichael G Fried
Mar 1, 2006·International Journal of Biological Macromolecules·Aline TiratLukas Leder
Jan 29, 2003·Biochemistry·Joseph J RasimasAnthony E Pegg

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