NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS
Human DNA Polymerase β Deoxyribose Phosphate Lyase: SUBSTRATE SPECIFICITY AND CATALYTIC MECHANISM*

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DNA polymerase β (β-pol) cleaves the sugar-phosphate bond 3′ to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5′-incised by AP endonuclease, resulting in a 5′ 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of β-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of β-pol are similar. However, k cat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5′ dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of β-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5′-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.

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On sabbatical from the Department of Biology, Northeastern University, Boston, MA 02115.