Sequences that have the capacity to adopt alternative (i.e. non-
B) DNA structures in the human genome have been implicated in stimulating
genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (
H-DNA) caused genetic instability in mammals largely in the form of
DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing
H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular
triplex DNA in vitro. Herein, we used a mutation-reporter system containing
H-DNA to examine the relevance of DHX9 activity on naturally occurring
H-DNA structures in human cells. We found that
H-DNA significantly increased mutagenesis in small-interfering
siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with
H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of
H-DNA, we performed binding assays in vitro and
chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized
H-DNA, as evidenced by its binding to the
H-DNA structure and enrichment at the
H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing
H-DNA structures in vivo and support its role in the overall maintenance of
genomic stability at sites of alternatively structured
DNA.