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Crystal structures of human APOBEC3A and a chimera of APOBEC3B and APOBEC3A bound to ssDNA reveal an unanticipated ‘U-shaped’ binding mode and provide insight into target selectivity. APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5′-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

DMRT transcription factors, key regulators of metazoan sexual development, use a unique DNA binding mode critical for male-to-female sex reversal in humans. DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo . We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

DNA-repair enzyme Tdp2 hydrolyzes the 5'-phosphotyrosine bond formed by topoisomerases and is associated with resistance to anticancer drugs that trap such complexes. The crystal structures of zebrafish Tdp2 bound to DNA offer insight into substrate recognition. In addition, the crystal structure of nematode Tdp2 suggests a potential mechanism for autoregulation. The DNA-repair enzyme Tdp2 resolves 5′-phosphotyrosyl DNA adducts and mediates resistance to anticancer drugs that target covalent topoisomerase–DNA complexes. Tdp2 also participates in key signaling pathways during development and tumorigenesis and cleaves a protein-RNA linkage during picornavirus replication. The crystal structure of zebrafish Tdp2 bound to DNA reveals a deep, narrow basic groove that selectively accommodates the 5′ end of single-stranded DNA in a stretched conformation. The crystal structure of the full-length Caenorhabditis elegans Tdp2 shows that this groove can also accommodate an acidic peptide stretch in vitro , with glutamate and aspartate side chains occupying the DNA backbone phosphate–binding sites. This extensive molecular mimicry suggests a potential mechanism for autoregulation and interaction of Tdp2 with phosphorylated proteins in signaling. Our study provides a framework to interrogate functions of Tdp2 and develop inhibitors for chemotherapeutic and antiviral applications.

The APOBEC3B (A3B) single-stranded DNA (ssDNA) cytosine deaminase has important roles in innate immunity but is also a major endogenous source of mutations in cancer. Previous structural studies showed that the C-terminal catalytic domain of human A3B has a tightly closed active site, and rearrangement of the surrounding loops is required for binding to substrate ssDNA. Here we report structures of the A3B catalytic domain in a new crystal form that show alternative, yet still closed, conformations of active site loops. All-atom molecular dynamics simulations support the dynamic behavior of active site loops and recapitulate the distinct modes of interactions that maintain a closed active site. Replacing segments of A3B loop 1 to mimic the more potent cytoplasmic deaminase APOBEC3A leads to elevated ssDNA deaminase activity, likely by facilitating opening of the active site. These data collectively suggest that conformational equilibrium of the A3B active site loops, skewed toward being closed, controls enzymatic activity by regulating binding to ssDNA substrates.