Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases

Abstract

Mitochondrial DNA polymerase ␥ (pol ␥) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5 side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol ␥ acts to catalyze the removal of a 5-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol ␥ is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH 4 . For both DNA pol ␥ and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol ␤, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA. Abasic (AP)1 sites in DNA are produced frequently by spontaneous base loss or by the action of DNA glycosylases that initiate base excision repair of damaged bases in DNA (1, 2). If AP sites are not repaired quickly, DNA polymerases are capable of replicating through these non-instructional lesions, resulting in frequent misincorporation. Cells have adapted vigorous mechanisms for the repair of AP sites, usually beginning with incision of the phosphodiester backbone on the 5Ј side of the lesion by AP endonuclease to produce a 3Ј-OH terminus adjacent to a 5Ј-deoxyribose phosphate, or 5Ј-dRP group (3, 4). The 3Ј-OH terminus provides a primer for repair synthesis by DNA polymerase. The 5Ј-dRP moiety must be removed in the course of repair. This is frequently accomplished by a ␤-elimination reaction catalyzed by an AP lyase activity. When an AP lyase acts on an exposed 5Ј-dRP residue produced by a class II AP endonuclease, the activity may be considered as a dRP lyase. The dRP lyase mechanism involves nucleophilic attack on the C-1 position of the 5Ј-dRP group by a free amino group of the enzyme (5). This produces a transient covalent intermediate in which the DNA substrate is linked to the enzyme as a Schiff base that can be stabilized by treatment with strong reducing agents, such as sodium borohydride. This borohydride trapping reaction has been used to identify a number of DNA repair enzymes with AP lyase activity, including several DNA glycosylases (6, 7).Eukaryotic cells have redundant pathways for repairing AP sites in nuclear DNA. Under most circumstances, AP sites are repaired by a pathway that employs the dedicated repair polymerase, DNA pol ␤. Recently, Matsumoto and Kim (8) showed that the pol ␤ is especially well adapted to function in base excision repair, since it contains a dRP lyase activity in a small domain not required for polymerase activity. The active site of the pol ␤ dRP lyase has been localized to a heli...

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

“…Our finding that UL30 possesses AP and 5Ј dRP lyase activities adds to the list of activities already associated with this enzyme, namely DNA Pol, 3Ј-5Ј proofreading exonuclease and RNaseH activities (4). In this regard, UL30 resembles the mitochondrial replicase Pol ␥, which, apart from its Pol activity, also exhibits proofreading exonuclease and 5Ј dRP lyase activities (16,(19)(20)(21). Although cellular organisms have evolved to encode specialized DNA repair enzymes, there are several examples where multiple tasks are performed by a small set of enzymes.…”

“…In this case Pol ␥, with its class IIa aminoacyl tRNA synthetase-type processivity factor, TWINKLE helicase, and mitochondrial ssDNA binding protein mediate mitochondrial DNA replication (21). However, Pol ␥ also plays a primary role in mitochondrial genome maintenance via BER, where it is required for repair synthesis, and as a DNA lyase (16,19). Similarly, in HSV-1, the seven essential replication factors perform DNA replication functions but have also been shown to directly mediate recombination and DNA repair reactions as illustrated by the recombination functions of ICP8 (22,23) and the DNA lyase activity of UL30 described herein.…”

Protection of INS-1 Cells From Free Fatty Acid–Induced Apoptosis by Targeting hOGG1 to Mitochondria

“…These enzymes have been characterized by Pinz and Bogenhagen (41) and are composed of an apurinic-endonuclease, which cleaves the phosphate backbone on the 5Ј side of the abasic site, ␥-polymerase, which attaches the new base to the 3ЈOH group and has lyase activity to remove the 5Ј-deoxyribose phosphate and a ligase, which reseals the phosphate backbone. However, in contrast to ␤-polymerase, the base excision repair polymerase in the nucleus, which has strong lyase activity, ␥-polymerase has very weak lyase activity (42). This renders mitochondria more sensitive to the excessive generation of abasic sites, which occurs when N 2 O 3 is formed by the reaction of NO with O 2 .…”

The Mitochondrial DNA Polymerase β from Crithidia fasciculata Has 5′-Deoxyribose Phosphate (dRP) Lyase Activity but Is Deficient in the Release of dRP

“…Notably, neither of these residues is conserved in the C. fasciculata enzyme. It is interesting that a similar pattern of E⅐dRP accumulation is observed for pol ␥ from Xenopus laevis (34), although a homologous dRP lyase domain has not been reported for that enzyme.…”