Abstract
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.
Highlights
The integrity of genomic DNA is challenged by various insults within the cell, including base modifications by exogenous and endogenous sources, unscheduled cleavage at non-standard recognition sequences [1], strand breaks and expansions and deletions induced by non-canonical DNA structures that can form transiently during replication and transcription [2,3,4,5,6]
We found that DHX9 recognizes H-DNA in the context of supercoiled plasmids, thereby protecting the structure from mung bean nuclease (MBN) cleavage, and that siRNA-mediated DHX9 depletion increased the frequencies of mutations induced by H-DNA, with deletions being mostly affected
We first determined whether DHX9 had the capacity to recognize H-DNA structures in plasmids because, unlike synthetic inter-molecular triplex DNA, its formation requires negative supercoiling and unwinding of duplex DNA
Summary
The integrity of genomic DNA is challenged by various insults within the cell, including base modifications by exogenous and endogenous sources, unscheduled cleavage at non-standard recognition sequences [1], strand breaks and expansions and deletions induced by non-canonical (i.e. non-B) DNA structures that can form transiently during replication and transcription [2,3,4,5,6]. Most repetitive DNA motifs, including short tandem repeats (microsatellites), inverted repeats, alternating purine-pyrimidine tracts, runs of purines pyrimidines (PuPy) and G-rich sequences, have the capacity to adopt alternatively structured DNA (non-B DNA structures), including slipped and looped-out conformations, cruciforms, left-handed Z-DNA, triplex (H-DNA) and quadruplex structures [7,8].
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