NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein.
Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.
Zharkov
D O
DO
Department of Pharmacological Sciences, The State University of New York, Stony Brook, New York 11794-8651, USA.
Rieger
R A
RA
Iden
C R
CR
Grollman
A P
AP
D000595:Amino Acid Sequence; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043382:DNA-Formamidopyrimidine Glycosylase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011392:Proline; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase