Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry.
Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.
Rieger
R A
RA
Department of Pharmacological Sciences, SUNY-Stony Brook, New York 11794-3400, USA.
McTigue
M M
MM
Kycia
J H
JH
Gerchman
S E
SE
Grollman
A P
AP
Iden
C R
CR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry*; D006018:Glycols / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D013936:Thymidine / Q000737:chemistry; D014357:Trypsin
D000596:Amino Acids; D003432:Cross-Linking Reagents; D006018:Glycols; D007202:Indicators and Reagents; D009841:Oligonucleotides; D010455:Peptides; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D014357:Trypsin; D013936:Thymidine