Insight into mechanisms of 3′-5′ exonuclease activity and removal of bulky 8,5′-cyclopurine adducts by apurinic/apyrimidinic endonucleases

Abstract

8,5′-cyclo-2'-deoxyadenosine (cdA) and 8,5′-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases-Escherichia coli Xth and human APE1-can remove 5′S cdA (S-cdA) at 3′ termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3′ end. To understand the structural basis of 3′ repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2′-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3′-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3′→5′ exonuclease function and its inhibition by 3′-terminal S-cdA residue. This structure was used as a template to model 3′-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3′ repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3′ DNA termini in E. coli and human cells, respectively.oxidative DNA damage | endonuclease IV | DNA glycosylase | base excision repair | damage specific endonuclease O xidative damage to DNA caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. If unrepaired, the damage will tend to accumulate and lead to premature aging, neurodegenerative disorders, and cancer (1). More than 80 different oxidative modifications of DNA bases and sugar backbone have been identified to date (2). Diastereoisomeric (5′S)-and (5′R)-8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) are generated by endogenous oxidative stress and ionizing radiation among other oxidized bases (Fig. 1A). 8,5′-cyclo-2′-deoxypurines (cdPu) are generated by hydroxyl radical attack at C5′ sugar by H-abstraction resulting in formation of C5′-centered sugar radical, which then reacts in the absence of oxygen with the C8 of the purine. Subsequent oxidation of the resulting N7-centered radical leads to intramolecular cyclization with the formation of a covalent bond between the C5′-and C8-positions of the purine nucleoside. When present in DNA duplex cdA causes large changes in backbone torsion angles, which leads to weakening of base pair hydrogen bonds and strong perturbations of the helix conformation near the lesion for both diastereoisomers. Interestingly, the glycosidic bond in S-cdA is approximately 40-fold more resistant to acid hydrolysis compared with regular dA, implying that this base lesion wou...

A unique DNA-binding mode of African swine fever virus AP endonuclease

“…However, the orientations of the outer α-helices of AsfvAP are significantly different from those of BaNfo, MtEn-doIV, and TtNfo; the root mean square deviation (rmsd) values between AsfvAP and the three homologous structures are all greater than 4.0 Å. In addition to apo-structure, several complex structures of EcNfo have also been reported previously 29,30 . Compared to AsfvAP, the overall conformation of EcNfo is more similar to those of BaNfo, MtEndoIV, and TtNfo, might due to the higher sequence identities between them (Fig.…”

Unveiling the non-repair face of the Base Excision Repair pathway in RNA processing: A missing link between DNA repair and gene expression?

“…In this way, the NIR action avoids the generation of potentially toxic AP-intermediates [28]. Several uncanonical substrates are processed by APE1 through NIR activity, including 5,6-dihydro-2′-deoxyuridine (DHU) [20,28], 5,6-dihydrothymidine (DHT) [20,28], 5-hydroxy-2′-deoxyuridine (5OHU) [20,28], 5-hydroxy-2′-deoxycitidine (5OHC) alpha-2′-deoxynucleosides (αdA, αdT and αdC) [20,[28][29][30][31], the majority of which are generated under ionizing radiation (IR) and exposure to certain drugs. This non-canonical activity of APE1 could explain how, on the contrary to what is observed in APE1 deficient cells [32][33][34], the lack of DNA glycosylases does not sensitize cells or mice to oxidative agents and IR [35][36][37].…”

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once

“…Given the small binding pocket, the mechanism for how Nfo and APE1 can accommodate a damaged base in nucleotide incision repair is still unclear, although some suggestion is given by a structure with a complete nucleotide at the active site of APE1 [76]. Another clue is given by another enzyme that structurally resembles APE1, TDP2 that incise 5′ tyrosyl groups from ssDNA and RNA.…”