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ExoChew-Seq: An in vitro method for investigating singlestranded DNA-protein interactions

ABSTRACT: During various cellular DNA transactions such as DNA replication and repair, genomic doublestranded DNA (dsDNA) is transiently unwound to generate single-stranded DNA (ssDNA). Several proteins interact with ssDNA, site- and/or structure-specifically, to regulate key cellular processes. Investigating these processes requires genome-wide identification of these ssDNA sequences. However, because of the transient nature of ssDNA in vivo techniques cannot reveal all such sequences requiring an in vitro alternative. Traditionally, ssDNA libraries are prepared by boiling or alkali-treatment of dsDNA libraries, but these techniques have their limitations as ssDNA can rapidly reanneal to generate dsDNA. We present here ExoChew, a novel enzymatic technique, which generates persistent ssDNA libraries from dsDNA libraries. In ExoChew, T7 exonuclease (T7 exo) or E. coli exonuclease III (Exo III), which binds and cleaves dsDNA in a specific direction, generates ssDNA libraries from dsDNA libraries. To validate the technique and demonstrate its applications, we conducted a genome-wide search for hnRNP K binding sites in an ExoChew-generated ssDNA library from mouse genomic DNA. The hnRNP K-bound ssDNA fragments were sequenced (ExoChew-seq) and a consensus hnRNP K binding motif was identified. Two other potential uses of ExoChew are the generation and genome-wide identification of ssDNA secondary structures and protein-ssDNA interactions.

ORGANISM(S): Mus musculus Escherichia coli

PROVIDER: GSE308562 | GEO | 2025/09/19

REPOSITORIES: GEO

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