Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q

Abstract

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5′ of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ–DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.

Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA

“…1, 2). Similar base-ipping mechanisms have been observed for various nucleic acid repair or modifying enzymes, including DNA glycosylase, cytosine methyltransferase, dsRNA adenosine deaminase, and lesion-speci c endonuclease [25][26][27][28][29][30][31][32] . A hallmark feature of these enzymes is the intercalation of an amino acid side chain into DNA/RNA base stacks to ll a void in the double helix 33 .…”

Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases

“…The enzyme binds three Zn 2+ ions; two of them reside in the PHP domain and are directly involved in phosphodiester bond hydrolysis, while the third Zn 2+ is found in a separate Zn-binding domain [ 167 ]. More recently, the crystal structures of PfuEndoQ bound to dU-, dI-, and AP site-containing DNA duplexes were solved ( Figure 4 B) [ 172 ]. The highest-resolution structure of PfuEndoQ with the U:G substrate revealed an active site with a single Zn 2+ ion coordinated by Glu76, His84, His139, and the oxygen atom of the scissile phosphodiester bond [ 172 ].…”

“…More recently, the crystal structures of PfuEndoQ bound to dU-, dI-, and AP site-containing DNA duplexes were solved ( Figure 4 B) [ 172 ]. The highest-resolution structure of PfuEndoQ with the U:G substrate revealed an active site with a single Zn 2+ ion coordinated by Glu76, His84, His139, and the oxygen atom of the scissile phosphodiester bond [ 172 ]. The E76A, H84A, and H139A single amino acid substitutions completely abolish the endonucleolytic activity of EndoQ, suggesting that this Zn 2+ is essential for phosphodiester bond cleavage [ 167 ].…”

Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea

“…Recently, the crystal structures of P. furiosus EndoQ bound to DNA substrates containing U, Hx, or an AP lesion have been solved ( Shi et al, 2021 ), demonstrating the mechanisms of recognition and cleavage of deaminated bases in DNA by this endonuclease. The structures of the P. furiosus EndoQ-DNA complexes show that a deep active-site pocket in the enzyme would be shaped by a concerted swing motion of its zinc-binding and C-terminal helical domains ( Shi et al, 2021 ), which allows the extruded deaminated bases to be accommodated. Furthermore, U and Hx bases associate with amino acid residues in this pocket, which is coordinated with an essential magnesium ion ( Shi et al, 2021 ).…”

“…The structures of the P. furiosus EndoQ-DNA complexes show that a deep active-site pocket in the enzyme would be shaped by a concerted swing motion of its zinc-binding and C-terminal helical domains ( Shi et al, 2021 ), which allows the extruded deaminated bases to be accommodated. Furthermore, U and Hx bases associate with amino acid residues in this pocket, which is coordinated with an essential magnesium ion ( Shi et al, 2021 ). Thus, the EndoQ-DNA complex structures provide mechanistic insights into damaged DNA recognition and cleavage by this endonuclease, which is helpful for understanding how EndoQ recognizes and cleaves these structurally diverse damaged DNA substrates rather than undamaged DNA substrate.…”