Study of the Lyase Activity of Human DNA Polymerase β Using Analogues of the Intermediate Schiff Base Complex

Sasha M. Daskalova, Xiaoguang Bai, Sidney Hecht

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

DNA polymerase β (Pol β) participates in mammalian base excision repair. The enzyme has a two-domain architecture, reflecting its dual functionality. The polymerase activity, which replaces damaged nucleosides removed during an initial excision process, is within the C-terminal 31 kDa domain, while the N-terminal 8 kDa domain participates in a lyase function, working to remove a 5′-deoxyribose phosphate (5′-dRP) moiety from the damaged DNA substrate. The currently accepted mechanism of the lyase reaction involves a transient covalent enzyme-DNA intermediate in the form of a Schiff base connecting Lys72 of the enzyme with the 5′-dRP moiety. The Schiff base intermediate is resolved via a β-elimination reaction, initiated by abstraction of a C2′-H atom from the 5′-dRP moiety. Presently, we describe the preparation of three Pol β enzymes modified at position 72 with aminooxy or hydrazinyl analogues of lysine. These enzymes form transient covalent bonds with the 5′-dRP moiety of the damaged DNA, in the form of an oxime or hydrazone, respectively. Both types of enzyme DNA intermediates are ultimately resolved by the lyase activities of each of the modified enzymes. Unsurprisingly, the formation and resolution of these E-S complexes proceed with diminished kinetics, and with an altered pH profile. The performed experiments provide additional support for Schiff base formation as an obligatory intermediate on the pathway to DNA repair, as well as for the proposed participation of Lys72 in effecting opening of the 5′-dRP ring via protonation of the ring oxygen atom, and for complex resolution via a β-elimination reaction.

Original languageEnglish (US)
Pages (from-to)2711-2722
Number of pages12
JournalBiochemistry
Volume57
Issue number18
DOIs
StatePublished - May 8 2018

ASJC Scopus subject areas

  • Biochemistry

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