Characterization of a promiscuous DNA sulfur binding domain and application in site-directed RNA base editing

Abstract

Phosphorothioate (PT)-modification was discovered in prokaryotes and is involved in many biological functions such as restriction-modification systems. PT-modification can be recognized by the sulfur binding domains (SBDs) of PT-dependent restriction endonucleases, through coordination with the sulfur atom, accompanied by interactions with the DNA backbone and bases. The unique characteristics of PT recognition endow SBDs with the potential to be developed into gene-targeting tools, but previously reported SBDs display sequence-specificity for PT-DNA, which limits their applications. In this work, we identified a novel sequence-promiscuous SBDHga from Hahella ganghwensis. We solved the crystal structure of SBDHga complexed with PT-DNA substrate to 1.8 Å resolution and revealed the recognition mechanism. A shorter L4 loop of SBDHga interacts with the DNA backbone, in contrast with previously reported SBDs, which interact with DNA bases. Furthermore, we explored the feasibility of using SBDHga and a PT-oligonucleotide as targeting tools for site-directed adenosine-to-inosine (A-to-I) RNA editing. A GFP non-sense mutant RNA was repaired at about 60% by harnessing a chimeric SBD-hADAR2DD (deaminase domain of human adenosine deaminase acting on RNA), comparable with currently available RNA editing techniques. This work provides insights into understanding the mechanism of sequence-specificity for SBDs and for developing new tools for gene therapy.

The binding affinity‐dependent inhibition of cell growth and viability by DNA sulfur‐binding domains

“…The growth of PT + strains with Hga and Gsu was completely suppressed (Figure 3b, Table 1). However, the mutant Hga-H25A, which has minimal binding activity for PT-DNA (Hu et al, 2023), did not generate a detectable suppressive effect on the PT + host (Figure 3c). Taken together, the prerequisite for the lethal growth phenotype is the simultaneous binding of a full PT site by two SBD molecules.…”

“…Therefore, we wanted to test SBDs with varied binding affinities for PT-DNA to examine their inhibitory effects on the growth of PT + cells. SBDs bind PT-DNA by embedding the sulfur atom into a hydrophobic surface cavity, and interacting with phosphate groups and bases adjacent to the PT site by hydrogen bonds and electrostatic interactions (Hu et al, 2023;Liu et al, 2018;Yu et al, 2020). To this end, three putative SBDs that are linked with domains other than the HNH motif (Figure S4a) were mined using the method by Hu et al (2023) and based on the conservation of a sulfur atom coordination motif (blue filled triangles in Figure S4c) and adjacent amino acid residues (blue and red rectangles, Figure S4c).…”

“…SBDs bind PT-DNA by embedding the sulfur atom into a hydrophobic surface cavity, and interacting with phosphate groups and bases adjacent to the PT site by hydrogen bonds and electrostatic interactions (Hu et al, 2023;Liu et al, 2018;Yu et al, 2020). To this end, three putative SBDs that are linked with domains other than the HNH motif (Figure S4a) were mined using the method by Hu et al (2023) and based on the conservation of a sulfur atom coordination motif (blue filled triangles in Figure S4c) and adjacent amino acid residues (blue and red rectangles, Figure S4c). Along with the two SBD-HNH homologs (SprMcrA and GsuMcrA), the three full-length SBD-containing proteins were examined using the transformation efficiency assay (Hu et al, 2023;Yu et al, 2018) for their restrictive activity on PT-DNA modified by the dnd gene cluster from S. enterica Cerro 87, which governs phosphorothioation of 5′-G PS AAC-3′/5′-G PS TTC-3′.…”

“…In addition to methylation-dependent restriction enzymes, over 2000 genes for putative PT-dependent restriction enzymes (PDREs) have been discovered in the sequenced bacterial genomes (Hu et al, 2023;Liu et al, 2018;Rakesh et al, 2024;Wang et al, 2019).…”