DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination.

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

New insight into the biology of R-loops.

R-loops form when RNA hybridizes with its template DNA generating a three-stranded structure leaving a displaced single strand non-template DNA. During transcription negative supercoiling of DNA behind the advancing RNA polymerase will facilitate the formation of R-loops by the nascent RNA as the DNA is under wound to facilitate transcription. In theory R-loops are classified into pathological and non-pathological depending on the context of its formation. R-loop which are formed normally in various physiological events like in gene regulation and at immunoglobulin class switch regions are considered non-pathological, whereas abnormally stable R-loop which leads to genomic instability are considered pathological. Although pathological R-loop formation is a rare event but once formed completely blocks transcription, mRNA export, elevates mutagenesis, and inhibits gene expression. Hence, R-loop either prevents or induces genomic instability indirectly and are potentially an endogenous source of DNA lesion. Although the existence of R-loop has been reported few decades ago, but only recently we have gained knowledge about its formation and resolution in cells due to the availability of reagents. R-loop biology has generated immense interest in past few years since it connects the important biological processes such as transcription, mRNA splicing, DNA replication, recombination and repair. In this review I will focus on the recent progress made about formation and resolution of R-loop, based on the methodologies that are currently available to study R-loop using biochemical, cell biology and molecular biology approaches.

DHX9 helicase promotes R-loop formation in cells with impaired RNA splicing.

R-loops are stable nucleic acid structures that have important physiological functions, but which also pose a significant threat to genomic stability. Increased R-loops cause replication stress and chromosome fragility and have been associated with diseases such as neurodegeneration and cancer. Although excessive R-loops are a feature of cells that are defective in RNA processing, what causes them to form is unclear. Here, we demonstrate that DHX9 (RNA helicase A) promotes the formation of pathological and non-pathological R-loops. In the absence of splicing factors, formation of R-loops correlates with the prolonged association of DHX9 with RNA Polymerase II (RNA Pol II). This leads to the production of DNA-RNA hybrid, which traps RNA Pol II on chromatin with the potential to block DNA replication. Our data provide a molecular mechanism for the formation of R-loops that is relevant to neurodegenerative diseases and cancers in which deregulated RNA processing is a feature.

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells.

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc(-)) DT40 cells arrest in G2 phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc(-) cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G2 checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G2 arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G2 phase.

Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes.

Human DHX9 helicase, also known as nuclear DNA helicase II (NDH II) and RNA helicase A (RHA), belongs to the SF2 superfamily of nucleic acid unwinding enzymes. DHX9 melts simple DNA-DNA, RNA-RNA, and DNA-RNA strands with a 3'-5' polarity; despite this little is known about its substrate specificity. Here, we used partial duplex DNA consisting of M13mp18 DNA and oligonucleotide-based replication and recombination intermediates. We show that DHX9 unwinds DNA- and RNA-containing forks, DNA- and RNA-containing displacement loops (D- and R-loops), and also G-quadruplexes. With these substrates, DHX9 behaved similarly as the RecQ helicase WRN. In contrast to WRN, DHX9 melted RNA-hybrids considerably faster than the corresponding DNA-DNA strands. DHX9 preferably unwound R-loops and DNA-based G-quadruplexes indicating that these structures may be biologically relevant. DHX9 also unwound RNA-based G-quadruplexes that have been reported to occur in human transcripts. It is believed that an improper dissolution of co-transcriptionally formed D-loops, R-loops, and DNA- or RNA-based G-quadruplexes represent potential roadblocks for transcription and thereby enhance transcription associated recombination events. By unwinding these structures, DHX9 may significantly contribute to transcriptional activation and also to the maintenance of genomic stability.

Human DHX9 helicase unwinds triple-helical DNA structures.

Naturally occurring poly(purine.pyrimidine) rich regions in the human genome are prone to adopting non-canonical DNA structures such as intramolecular triplexes (i.e., H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several disease-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3' --> 5' polarity with respect to the displaced third strand. Helicase activity required a 3'-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order reaction. In contrast, very little if any helicase activity was detected on blunt triplexes, triplexes with 5'-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3'-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures.

WRN helicase unwinds Okazaki fragment-like hybrids in a reaction stimulated by the human DHX9 helicase.

Mutations in the Werner gene promote the segmental progeroid Werner syndrome (WS) with increased genomic instability and cancer. The Werner gene encodes a DNA helicase (WRN) that can engage in direct protein-protein interactions with DHX9, also known as RNA helicase A or nuclear DNA helicase II, which represents an essential enzyme involved in transcription and DNA repair. By using several synthetic nucleic acid substrates we demonstrate that WRN preferably unwinds RNA-containing Okazaki fragment-like substrates suggesting a role in lagging strand maturation of DNA replication. In contrast, DHX9 preferably unwinds RNA-RNA and RNA-DNA substrates, but fails to unwind Okazaki fragment-like hybrids. We further show that the preferential unwinding of RNA-containing substrates by WRN is stimulated by DHX9 in vitro, both on Okazaki fragment-like hybrids and on RNA-containing 'chicken-foot' structures. Collectively, our results suggest that WRN and DHX9 may also cooperate in vivo, e.g. at ongoing and stalled replication forks. In the latter case, the cooperation between both helicases may serve to form and to dissolve Holliday junction-like intermediates of regressed replication forks.