Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed β-elimination at abasic sites

Andrew J. Kurtz, M. L. Dodson, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via β-elimination. An amine serves as the reactive nucleophile to attack C1′ on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed β-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision, N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal α-amino group, not an ε-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed β-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.

Original languageEnglish (US)
Pages (from-to)7054-7064
Number of pages11
JournalBiochemistry
Volume41
Issue number22
DOIs
StatePublished - Jun 4 2002
Externally publishedYes

Fingerprint

Nucleophiles
Peptides
Schiff Bases
DNA
lysyl-tryptophyl-alpha-lysine
Repair
Deoxyribose
Acetylation
Tryptophan
Sugars
DNA Repair
Alanine
Lysine
DNA Damage
Amines
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed β-elimination at abasic sites. / Kurtz, Andrew J.; Dodson, M. L.; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 41, No. 22, 04.06.2002, p. 7054-7064.

Research output: Contribution to journalArticle

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