| The etiology of cancer involves the accumulation of mutations that stem from the persistence of unrepaired DNA damage. DNA is under constant attack by environmental factors. DNA damage also occurs from endogenously produced metabolites and by spontaneous hydrolysis of intrinsically unstable bonds in DNA. To maintain genetic information, all organisms possess fundamental cellular processes to repair damaged DNA. Enzymes of the base excision repair (BER) pathway recognize and remove potentially mutagenic base lesions. In humans, BER is typically initiated by DNA glycosylases, which hydrolyze the glycosylic bonds of modified bases to generate apurinic/apyrimidinic (AP) sites in DNA. AP sites are also primary lesions that result from spontaneous base loss. AP endonuclease 1 (Ape1) subsequently initiates the repair of AP sites by hydrolyzing the 5′-phosphodiester bond of AP sites to create nicks with 3′-hydroxyl and 5′ -deoxyribose-5-phosphate (dRP) termini. Polβ catalyzes both DNA synthesis at the 3′-OH terminus and excision of the 5 ′-dRP moiety prior to completion of repair by DNA ligase. In addition to its AP endonuclease activity, Ape1 also possesses 3′ -phosphodiesterase, 3′-phosphatase, and 3′ → 5′ exonuclease functions specific for the 3′ termini of internal nicks and gaps.; This thesis investigated several coordinated enzymatic activities of Ape1 and Polβ in vitro. We demonstrated that the 3 ′ → 5′ exonuclease activity of Ape1 is active at both mismatched and non-mismatched 3′ termini. However, the activity is inhibited by the presence of adjacent 5′ -dRP groups, which are produced by the AP endonuclease activity of Ape1. Excision of the 5′-dRP by Polβ, or physical displacement of the 5′-incised abasic residue, alleviated the inhibition of the Ape1 exonuclease activity. Furthermore, tight binding of Ape1 to its 5′-incised AP site product results in the inhibition of DNA synthesis at the adjacent 3′-OH terminus by Polβ. This inhibitory effect was likewise abated by removal of the 5′-dRP. Ape1 can stimulate 5′-dRP excision by Polβ, by a mechanism independent of magnesium. We also identified a second stimulatory mechanism, independent of Ape1, by which 5′ -dRP excision is stimulated under conditions that support DNA synthesis. These results further establish the central roles of Ape1 and Polβ in coordinating mammalian BER. |
