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Review
. 2019 Sep;44(9):765-781.
doi: 10.1016/j.tibs.2019.04.006. Epub 2019 May 9.

Emerging Roles of DNA Glycosylases and the Base Excision Repair Pathway

Elwood A Mullins  1 , Alyssa A Rodriguez  1 , Noah P Bradley  1 , Brandt F Eichman  2
Affiliations
Review

Emerging Roles of DNA Glycosylases and the Base Excision Repair Pathway

Elwood A Mullins et al. Trends Biochem Sci. 2019 Sep.

Abstract

The base excision repair (BER) pathway historically has been associated with maintaining genome integrity by eliminating nucleobases with small chemical modifications. In the past several years, however, BER was found to play additional roles in genome maintenance and metabolism, including sequence-specific restriction modification and repair of bulky adducts and interstrand crosslinks. Central to this expanded biological utility are specialized DNA glycosylases - enzymes that selectively excise damaged, modified, or mismatched nucleobases. In this review we discuss the newly identified roles of the BER pathway and examine the structural and mechanistic features of the DNA glycosylases that enable these functions.

Keywords: DNA damage; DNA glycosylase; DNA repair; base excision repair; interstrand crosslink; secondary metabolite.

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Figures

Figure 1.
Figure 1.. Base Excision Repair of Damaged Nucleobases.
(A) Common DNA lesions resulting from alkylation, oxidation, and deamination of nucleobases. 3mA, 3-methyl-2’-deoxyadenosine; 7mG, 7-methyl-2’-deoxyguanosine; 8oxoG, 8-oxo-2’-deoxyguanosine; εA, 1,N6-etheno-2’-deoxyadenosine; mFapyG, methyl derivative of N6-(2’-deoxyribosyl)-2,6-diamino-4-oxo-5-formamidopyrimidine (FapyG); dU, 2’-deoxyuridine. (B) General steps in the base excision repair (BER) pathway. BER is initiated by lesion-specific DNA glycosylases, which remove damaged nucleobases to create an apurinic/apyrimidinic (AP) site. An AP endonuclease (or a bifunctional DNA glycosylase) then incises the modified strand, producing a single-strand break. As necessary, the break is processed by one of several enzymes to create a gap with a 3’-hydroxyl group and a 5’-phosphoryl group. A DNA polymerase fills the gap with new DNA, and a DNA ligase seals the strand to complete repair. In eukaryotes, if strand incision is performed by an AP endonuclease, repair synthesis occurs before end processing, displacing the AP site.
Figure 2.
Figure 2.. Initiation of Base Excision Repair by DNA Glycosylases.
(A) Base excision and strand incision reactions performed by monofunctional and bifunctional DNA glycosylases. Monofunctional enzymes catalyze only base excision, wherein the glycosidic bond between the nucleobase and the phosphodeoxyribose backbone is hydrolyzed, removing the nucleobase and creating an AP site. Bifunctional enzymes catalyze both base excision and strand incision (lyase activity). During removal of the nucleobase, most bifunctional glycosylases form an iminium intermediate, which covalently links the protein and the DNA. Some bifunctional enzymes, however, initially hydrolyze the glycosidic bond to create an AP site before then converting the AP site to an iminium intermediate. Following base excision, all bifunctional DNA glycosylases incise the strand on the 3’-side of the AP site (β-elimination), generating a single-strand break with a 3’-phospho-α,β-unsaturated aldehyde (PUA) group and a 5’-phosphoryl group. Some bifunctional enzymes also subsequently incise the strand on the 5’-side of the PUA (δ-elimination), leaving a 3’-phosphate, which must also be removed prior to repair synthesis, requiring the phosphatase activity of a separate enzyme. Alternatively, if β-elimination occurs following strand incision by an AP endonuclease, a gap is generated with the 3’-hydroxyl and 5’-phosphoryl groups necessary for synthesis and ligation. (B) X-ray crystal structures of the bifunctional DNA glycosylase Fpg (green) bound to DNA (orange and yellow) containing an 8oxoG lesion (red; PDB ID: 3GO8, 3GPY) [82]. After base flipping, Met77, Arg112, and Phe114 (not shown) fill the void in the duplex. (C) An extrahelical 8oxoG lesion in the nucleobase binding pocket of Fpg. Hydrogen-bonding interactions are indicated with dashed lines.
Figure 3.
Figure 3.. Removal of Bulky Adducts by AlkD.
(A) Bulky adducts excised by AlkD. 7POBG, 7-pyridyloxobutyl-2’-deoxyguanosine; O2-POBC, O2-pyridyloxobutyl-2’-deoxycytidine; YTMA, 3-yatakemycinyl-2’-deoxyadenosine. AlkD eliminates bulky lesions with modifications located in either the major (7POBG) or the minor (O2-POBC and YTMA) groove. (B,D) X-ray crystal structure of AlkD (cyan) in complex with free 3-methyladenine (3mAde) nucleobase (purple) and DNA (orange and yellow) containing a tetrahydrofuran (THF) spacer (purple) to mimic an AP site (PDB ID: 5CLE) [29]. Water molecules located in the large cavity between the protein and the DNA are depicted as red spheres. (C,E) X-ray crystal structure of AlkD in complex with an excised yatakeymycinyladenine (YTMAde) nucleobase (purple) and DNA containing an AP site (purple; PDB ID: 5UUF) [26]. Hydrogen-bonding interactions are indicated with dashed lines. Unlike DNA glycosylases that use a traditional base-flipping mechanism, the catalytic residues (Trp109, Asp113, and Trp187) present in AlkD are located on the protein surface, not recessed in a nucleobase binding pocket. Without a catalytic requirement for base flipping, the lack of protein-DNA contacts in the major groove and the large solvent-filled cavity between the protein and the minor groove allow AlkD to recognize and excise nucleobases with bulky modifications at any position.
Figure 4.
Figure 4.. Unhooking of Interstrand Crosslinks by AlkZ.
(A) Interstrand crosslink (ICL) formed between two 2’-deoxyguanosine nucleotides by azinomycin B (AZB). AZB preferentially reacts in GNC sequences to form {1–3} crosslinks. (B) Hypothetical models of AlkZ (green and yellow) bound to DNA (gray and blue) containing an AZB ICL (red). The models were constructed by rigid-body docking of an X-ray crystal structure of AlkZ (PDB ID: 5UUJ) [50] and a computationally derived model of DNA containing an AZB ICL [38]. The DNA could be docked equally well in either of two binding orientations by placing the β11/β12 hairpin into the minor groove across from the ICL. Each orientation positions one of the two modified nucleotides (G1 or G2) near Gln39 in the putative active site of AlkZ. (C,D) Hypothetical model of an AlkZ dimer bound to DNA containing an AZB ICL. Docking two molecules of AlkZ with a single AZB ICL creates a network of salt bridges (Glu152 and Arg153), hydrogen bonds (Ser304 and Arg308), and hydrophobic contacts (Ala309 and Pro340) at the protein interface. (E) Alternate mechanisms of AZB ICL processing by the BER pathway. Concerted unhooking of both strands, consistent with the hypothetical dimeric complex, produces two closely spaced AP sites, potentially leading to a double-strand break (DSB). Sequential unhooking and repair of each strand avoids the concurrence of multiple AP sites and minimizes the possibility of a DSB.
Figure 5.
Figure 5.. Excision of Bulky Adducts and Interstrand Crosslinks by NEIL1 and NEIL3.
(A) Bulky adducts and interstrand crosslinks removed by NEIL1 or NEIL3. NM-FapyG, nitrogen mustard derivative of FapyG; AFBrFapyG, aflatoxin B1 derivative of FapyG; PSO, psoralen. Both psoralen and AP sites form {1–2} crosslinks by modifying nucleobases on opposing strands. (B) Triplex substrate for NEIL1. The crosslink is colored red and nascent DNA produced by translesion synthesis is gray. Triplex structures are generated during replication-associated ICL repair (Supplemental Figure S1). (C) Convergent fork substrate for NEIL3. Nascent DNA generated during replication is colored gray. NEIL3 is recruited to ICLs after fork convergence and replisome ubiquitinylation (Supplemental Figure S1). (D) X-ray crystal structure of NEIL1 (cyan) bound to DNA (orange and yellow) containing a thymine glycol (Tg) lesion (red; PDB ID: 5ITY) [81]. Three intercalating residues (Met81, Arg118, and Phe120) stabilize the extrahelical conformation of the DNA substrate, while a flexible capping loop allows for accommodation and excision of bulky lesions. (E) Crystal structure of the glycosylase domain of NEIL3 (PDB ID: 3W0F) [73]. NEIL3 lacks the two intercalating residues that interact with the undamaged strand in the NEIL1 complex, as well as the flexible capping loop that contacts the Tg lesion. Consistent with a preference for non-duplex substrates, NEIL3 also lacks the basic residue (Arg274) that forms a salt bridge with the undamaged strand in the NEIL1 complex.
Figure 6.
Figure 6.. Sequence-Specific Excision of Adenine by R.Pabl.
(A) Double-strand break resulting from dual base excision and subsequent incision of AP sites on opposing strands. (B) X-ray crystal structure of tetrameric R.PabI (blue, green, and white) bound to non-specific DNA (orange and yellow; PDB ID: 5IFF) [94]. Four salt bridges formed by Arg70 and Asp71 are the only interactions between the two R.PabI dimers in the tetrameric search complex. (C) Crystal structure of dimeric R.PabI bound to specific DNA after dual excisions of adenine (Ade) nucleobase (red) to create AP sites (red) on opposing strands (PDB ID: 3WAZ) [90]. Binding of the recognition sequence induces a transition to a dimeric excision complex in which the GTAC base pairs are pulled apart and the void created in the duplex is stabilized by insertion of Gln155 and Arg156. (D) Hydrogen-bonding interactions in the dimeric product complex. DNA binding residues are colored according to protein subunit. R.PabI forms 13 sequence-specific and 21 non-sequence-specific hydrogen-bonding interactions with each strand in the palindromic product. (E) Recognition of Ade in the active site of R.PabI (stereodiagram). Hydrogen-bonding interactions are indicated with dashed lines. N6-methyl-2’-deoxyadenosine, N6-mA. Hydrogen bonds between Ade and backbone atoms in Ile66 and Val164 select for an N7-protonated substrate to catalyze excision. Sequence-specific methylation of Ade by M.PabI introduces steric clashes with these same backbone atoms to prevent catalytically productive binding and excision of N6-mA.
Figure I.
Figure I.. Base Excision by DNA Glycosylases.
(A) Putative mechanism of glycosidic bond cleavage. Addition of a water molecule or an amine group to the oxocarbenium intermediate would produce an AP site or an iminium crosslink, respectively. (B) Key catalytic residues required for base excision. Carboxylate (aspartate/glutamate) and carboxamide (asparagine/glutamine) functional groups are depicted both as lowest-energy resonance forms (left) and as resonance hybrids (right).
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