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.