Mapping of the 5'-2-deoxyribose-5-phosphate lyase active site in DNA polymerase beta by mass spectrometry.
The mechanism of the 5'-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA beta-polymerase (beta-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of beta-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys(72) residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys(72) as the sole Schiff base nucleophile in the 8-kDa domain of beta-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys(72) at the abasic site.
Deterding
L J
LJ
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Prasad
R
R
Mullen
G P
GP
Wilson
S H
SH
Tomer
K B
KB
D000595:Amino Acid Sequence; D001665:Binding Sites; D001894:Borohydrides; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D011487:Protein Conformation; D012545:Schiff Bases; D014357:Trypsin
D001894:Borohydrides; D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D012545:Schiff Bases; C025364:sodium borohydride; D004247:DNA; D019951:DNA Polymerase beta; D014357:Trypsin; D008239:Lysine