The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

Mullins, E.A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

doi:10.1038/nature15728

Cited by 52 publications

(102 citation statements)

References 59 publications

(69 reference statements)

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“…Based on these considerations, we speculate that replication fork convergence places ICLs in a single-stranded DNA context, allowing partial eversion of the cross-linked base into the active site of NEIL3, which is unusually open. As such, NEIL3’s mechanism is probably distinct from that of AlkD, a DNA glycosylase recently shown to cleave bulky base lesions in the complete absence of base eversion (Mullins et al, 2015). A recent report that Streptomyces orf1 DNA glycosylase can act on azinomycin B ICLs (Wang et al, 2016) suggests that ICL unhooking by DNA glycosylases is widely conserved in evolution.…”

Section: Discussionmentioning

confidence: 99%

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase

Semlow

Zhang

Budzowska

et al. 2016

Cell

170

237

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Summary During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved (“unhooked”) by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen- and abasic site-ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.

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Section: Discussionmentioning

confidence: 99%

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase

Semlow

Zhang

Budzowska

et al. 2016

Cell

170

237

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“…DNA glycosylases that excise alkylated DNA lesions often exhibit activity for a number of N3-and N7-methylated purines in addition to their preferred substrates (21,22). For example, Bacillus cereus AlkD displays robust activity for bulky minor groove N3-yatakemycinyldeoxyadenosine lesions, but also cleaves N3-methyldeoxyadenosine and N7-methyldeoxyguanosine (d7mG) (23,24). Therefore, we tested the ability of AlkZ to catalyze excision of N7-methylguanine (7mGua) from an oligonucleotide containing d7mG ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, we recently discovered a nonbase-flipping mechanism that enables glycosylase excision of bulky minor groove adducts and conceivably could enable ICL excision. The bacterial AlkD glycosylase is able to recognize and cleave deoxyadenosine adducts of the bulky natural product yatakemycin (YTM) without rotating the lesion from the duplex (24). Like AlkZ, AlkD adopts a C-shaped fold that engages DNA along the concave surface (22).…”

Section: Discussionmentioning

confidence: 99%

Structure of a DNA glycosylase that unhooks interstrand cross-links

Mullins

Warren

Bradley

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

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“…4,5 However, we recently showed that base flipping is not a requisite for catalysis, as demonstrated by the Bacillus cereus DNA glycosylase AlkD, which is specific for cationic lesions. 6 Time-resolved crystal structures of AlkD-mediated base excision revealed that the substrate nucleobase remains stacked in the duplex over the course of the reaction and that three active site residues—Asp113, Trp109, and Trp187—cradle the substrate deoxyribose without contacting the nucleobase itself. Importantly, the indole rings of both tryptophan side chains form a series of C-H/π interactions with the lesion deoxyribose, and computational modeling suggested that these interactions contribute to lowering the barrier to depurination.…”

mentioning

confidence: 99%

“…6,17 The active site was modeled from the side chains of Asp113, Trp109, and Trp187, truncated at their beta-carbons, and the lesion was modeled as the nucleoside (Supporting Information). To validate this model, we first calculated the activation energy to spontaneous depurination of d7mG.…”

mentioning

confidence: 99%

A Catalytic Role for C–H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD

et al. 2016

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DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually takes place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the CH/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair.

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The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

Cited by 52 publications

References 59 publications

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase

Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase

Structure of a DNA glycosylase that unhooks interstrand cross-links

A Catalytic Role for C–H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD

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