In Vitro Binding of GADD45A to RNA:DNA Hybrids

R-loops are three-stranded RNA:DNA hybrid structures that frequently form during transcription. While R-loop misregulation is associated with genome instability, cells also harness RNA-DNA hybrids in scheduled, "regulatory," R-loops to control gene expression. One regulatory role involves epigenetic gene regulation by the R-loop reader Growth Arrest and DNA Damage 45A (GADD45A). This small stress related protein promotes DNA demethylation by recruiting TET dioxygenase and Thymine DNA glycosylase to specific genomic loci. GADD45A requires adapters for its genomic localization. One such class of adapters are R-loops formed at certain CpG island promoters to which GADD45A binds directly, targets the demethylation machinery, and confers an open chromatin state. Here, we describe protocols for carrying out in vitro binding assays with GADD45A to RNA:DNA hybrids to biochemically study its direct binding to R-loops, specifically GADD45A pulldown and EMSA (electrophoretic mobility shift) assays.