Structure of a DNA glycosylase that unhooks interstrand cross-links

Abstract

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.

OUP accepted manuscript

“…( E ) Time-dependence of d7mG excision by AlkZ and YcaQ. The presence of a single product band in the AlkZ reaction is likely a result of EndoIV contamination in the work-up buffer, consistent with our previous analysis ( 20 ). ( F ) Structure and excision of mFaPy-dG DNA, treated with E. coli Fpg, YcaQ, or buffer for the specified time, followed by alkaline hydrolysis.…”

“…AZB displays potent antibacterial activity against a variety of bacterial species ( 7 ), antitumor activity at lower doses than mitomycin C ( 18 ), and initiates transcription of DNA damage response genes after treatment ( 19 ). The AZB synthesis cluster contains a novel DNA glycosylase, AlkZ, which cleaves AZB-ICLs and N 7-methylguanine monoadducts in vitro to produce an AP site that can be processed by the bacterial AP endonuclease EndoIV ( 14 , 20 ). AlkZ expression is induced during production of AZB, and cells that express alkZ are resistant to the cytotoxic effects of AZB, even across different bacterial species ( 14 ).…”

“…AlkZ belongs to the functionally uncharacterized HTH_42 superfamily of bacterial proteins known only for their arrangement of tandem winged helix-turn-helix (wHTH) motifs commonly found in transcription factors and other DNA binding proteins ( 21 ). The crystal structure of AlkZ revealed a novel fold in which three wHTH motifs scaffold a C-shaped protein with a positively charged, concave surface that contains several residues important for DNA glycosylase activity ( 20 ) ( Supplementary Figure S1 ). HTH_42 family proteins are widespread among antibiotic producers and human pathogens, including Escherichia coli , Clostridium difficile , and Staphylococcus aureus , among others ( 14 ).…”

Limited Capacity or Involvement of Excision Repair, Double-Strand Breaks, or Translesion Synthesis for Psoralen Cross-Link Repair in Escherichia coli

“…The bacteria additionally encodes a protein that confers self-resistance against this toxin (Wang et al 2016). Initial characterizations suggest the protein has activities that protect cells from forming cross-links, but could also potentially be involved in processing either monoadducts or cross-links when they form (Wang et al 2016;Mullins et al 2017). Further characterization of how this organism processes these lesions, as well as other compounds, will be important to determine how the lesions affect cell viability and genome integrity in humans and other organisms.…”

Induced‐Fit Recognition of CCG Trinucleotide Repeats by a Nickel–Chromomycin Complex Resulting in Large‐Scale DNA Deformation

“…This local instability is flexible enough to be able to adopt the geometrically optimal conformation that allows cytosines to extrude out to form the GGCC tetranucleotide patch compatible with specific binding mode of Ni II (Chro) 2 . Binding‐induced nucleotide extrusion has also been observed in structures of complexes between DNA repair proteins and DNA complexes . Other small molecules that target (CXG) n TNRs also induce nucleotide extrusion.…”