Modulation of the 5′-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase β by apurinic/apyrimidinic endonuclease 1

Abstract

The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Apel catalyzes hydrolysis of the 5′-phosphodiester bond of abasic DNA to create nicks flanked by 3′-hydroxyl and 5′-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) β catalyzes both DNA synthesis at the 3′-hydroxyl terminus and excision of the 5′-dRP moiety prior to completion of BER by DNA ligase. During BER, Apel recruits pol β to the incised apurinic/apyrimidinic site and stimulates 5′-dRP excision by pol β. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Apel to modulate the deoxynucleotidyltransferase and 5′-dRP lyase activities of pol β. We report here that Apel stimulates 5′-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Apel, in which conditions that support DNA synthesis by pol β also enhance 5′-dRP excision. Apel modulates the gap-filling activity of pol β by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Apel protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol β. However, this mutant protein retained the ability to stimulate 5′-dRP excision by pol β. Simultaneous monitoring of 5′-dRP excision and DNA synthesis by pol β demonstrated that the 5′-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.