Human DNA Polymerase β Deoxyribose Phosphate Lyase: SUBSTRATE SPECIFICITY AND CATALYTIC MECHANISM

Rajendra Prasad,William A Beard,Phyllis R Strauss,Samuel H Wilson

doi:10.1074/jbc.273.24.15263

Rajendra Prasad, William A Beard + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.24.15263

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Abstract

DNA polymerase beta (beta-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 beta-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 beta-pol are similar. However, kcat 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 beta-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.

Highlights

From the §Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ‡Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555
We investigated the catalytic efficiency and requirements of the ␤-pol deoxyribose 5-phosphate (dRP) lyase activity using intact AP site-containing DNA substrates or substrates that have been preincised with AP endonuclease
Since AP endonuclease incises the AP site DNA strand 5Ј to the AP site and ␤-pol incises 3Ј, the AP endonuclease DNA product bears a nick with a 5Ј-dRP moiety and a 3Ј-hydroxyl

Summary

SUBSTRATE SPECIFICITY AND CATALYTIC MECHANISM*

(Received for publication, February 6, 1998, and in revised form, April 1, 1998). Rajendra Prasad‡§, William A. 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 aminoterminal 8-kDa domain of ␤-pol and with the entire 39kDa polymerase. Matsumoto and Kim [32] demonstrated that ␤-pol catalyzes removal of dRP from AP endonuclease-incised AP sites via ␤-elimination, as opposed to hydrolysis, and that this dRP lyase activity resides in the amino-terminal 8-kDa domain of ␤-pol Further evidence that this reaction proceeds via ␤-elimination was obtained by Piersen et al [33], who showed that a Schiff base intermediate. We investigated the catalytic efficiency and requirements of the ␤-pol dRP lyase activity using intact AP site-containing DNA substrates or substrates that have been preincised with AP endonuclease. The findings are discussed in the context of the NMR and crystal structures of the ␤-pol 8-kDa domain and the requirements of ␤-elimination chemistry in the dRP lyase reaction

EXPERIMENTAL PROCEDURES

RESULTS

Intact AP site DNA substratee

DISCUSSION