Stimulation of 3'→5' exonuclease and 3'-phosphodiesterase activities of yeast Apn2 by proliferating cell nuclear antigen

The Apn2 protein of Saccharomyces cerevisiae contains 335 exonuclease and 3-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and twohybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNAbinding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 335 exonuclease and 3-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding. Cellular DNA is continually being damaged by a variety of extrinsic and intrinsic factors. Oxygen-free radicals formed during the course of normal cellular metabolism produce modified miscoding bases, abasic (AP) sites, and DNA strand breaks. AP sites are some of the most common lesions that arise in cellular DNA. It has been estimated that a mammalian cell loses up to 10,000 purines per day spontaneously from its genome (18). AP sites are also formed from the action of DNA glycosylases on modified bases. Oxidative attack on DNA also results in DNA strand breaks that have a 3-phosphate or 3-phosphoglycolate (3-PG) terminus (11, 13). The -lyase activity of some DNA glycosylases also leads to the formation of such modified 3 termini (28). Since these 3-end groups are refractory to DNA synthesis, their removal is an essential early step in the repair of these lesions (3). Class II AP endonucleases are multifunctional enzymes that function in the removal of AP sites as well as 3-blocking termini. Two families of class II AP endonuclease and repair diesterase enzymes have been identified. The endonuclease IV family contains endonuclease IV of Escherichia coli and the Apn1 protein, the major AP endonuclease of Saccharomyces cerevisiae. In addition to AP endonuclease activity, these enzymes possess 3-phosphatase and 3-phosphodiesterase activities that can remove 3-modified termini (22, 28). The exonuclease III (exoIII) family includes exoIII, the major AP endonuclease of E. coli, the Apn2 protein of S. cerevisiae, and Ape1, the dominant AP endonuclease in human cells. exoIII displays, in addition to the AP endonuclease activity, strong 335 exonuclease, 3-phosphodiesterase, and 3-phosphatase activities (28). Human Ape1, however, has a strong AP endonuclease activity but weak 335 exonuclease, 3-phosphodiesterase, and 3-phosphatase activities (23, 31). The Apn2 protein of S. cerevisiae exhibits a weak AP endonuclease activity and strong 335 exonuclease and 3-phosphodiesterase activities that are 30- to 40-fold more active than the AP endonuclease activity (25, 26).