Fetal hemoglobin (HbF, αγ) level is genetically controlled and modifies severity of adult hemoglobin (HbA, αβ) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.
The TraI protein of conjugative plasmid F factor binds and cleaves a single-stranded region of the plasmid prior to transfer to a recipient. TraI36, an N-terminal TraI fragment, binds ssDNA with a subnanomolar K(D) and remarkable sequence specificity. The structure of the TraI36 Y16F variant bound to ssDNA reveals specificity determinants, including a ssDNA intramolecular 3 base interaction and two pockets within the protein's binding cleft that accommodate bases in a knob-into-hole fashion. Mutagenesis results underscore the intricate design of the binding site, with the greatest effects resulting from substitutions for residues that both contact ssDNA and stabilize protein structure. The active site architecture suggests that the bound divalent cation, which is essential for catalysis, both positions the DNA by liganding two oxygens of the scissile phosphate and increases the partial positive charge on the phosphorus to enhance nucleophilic attack.
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