RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions

Chien, Yueh‐hsiu; Davidson, Norman

doi:10.1093/nar/5.5.1627

Cited by 32 publications

(19 citation statements)

References 22 publications

(20 reference statements)

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“…In line with this notion, we observed a significant increase in DNA:RNA hybrid formation in FANCM-depleted cells ( Figure 6 A; Figure S6 A). Importantly, purified FANCM was not only able to unwind replication fork structures, as shown previously ( Gari et al., 2008 ; Figure S6 B), but it efficiently unwound DNA:RNA hybrids in vitro ( Figures 6 B and 6C) despite such substrates being more stable than DNA:DNA hybrids found at a replication fork ( Chien and Davidson, 1978 ). The branch-migratable structures were designed to mimic both the 5′ and 3′ ends of a DNA:RNA hybrid, and our biochemical analyses have shown that FANCM can translocate along either the Watson or Crick strand in a 3′-5′ direction and disrupt DNA:RNA base pairing ( Figures 6 B and 6C).…”

Section: Resultssupporting

confidence: 81%

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription

et al. 2015

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SummaryDNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins.

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Section: Resultssupporting

confidence: 81%

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription

et al. 2015

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“…RNA:DNA hybrids are also formed during transcription, when a newly formed mRNA transcript is paired with one strand of duplex DNA (13). Notably, RNA:DNA hybrids have a relatively stable structure and withstand acidic conditions up to pH 5.5 (22,23). Several lines of evidence in this study indicated that RNA:DNA hybrids of bacterial origin play a key role in NLRP3 inflammasome activation by EHEC.…”

Section: Rnase H Deficiency In Bacteria Enhances Inflammasome Activatmentioning

confidence: 63%

Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome

Vanaja

Rathinam

Atianand

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance The nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome regulates capase-1-dependent maturation of interleukin-1β during infection with Gram-negative bacterial pathogens such as enterohemorrhagic Escherichia coli . Here we identified bacterial RNA:DNA hybrids as well as RNA as critical mediators of these responses. RNA:DNA hybrids and RNA gained access to the cytosol from phagolysosomal compartments during infection, leading to the assembly of NLRP3 inflammasome complex. Delivery of synthetic RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, whereas introduction of RNase H, which degrades hybrids, abolished inflammasome activation. Notably, an E. coli rnhA mutant, incapable of producing RNase H, induced elevated levels of NLRP3-dependent inflammasome activation. Collectively, these studies define bacterial RNA:DNA hybrids as a new microbe-associated molecular pattern with innate immune stimulatory activity during microbial infections.

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“…This is supported by GuHCl ability to increase the activity of Proteinase K and increase the torsional mobility of denatured proteins (at 1 M concentration) [ 54–56 ]. Furthermore, unlike SDS, chaotropes interacts with nucleic acids, altering their secondary and tertiary structure [ 57 , 58 ]. In fact, 1 M concentrations of GuHCl have been shown to reduce the Tm of DNA by 13°C, and increase the stringency of its hybridization, promoting correct base pairing [ 59 ].…”

Section: Discussionmentioning

confidence: 99%

Characterization of FFPE-induced bacterial DNA damage and development of a repair method

Bueso

Walker

Tangney

2020

Biology Methods and Protocols

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Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterised. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterise this FFPE-induced bacterial DNA damage, and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised - an optimised decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an in vitro reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts, and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.

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RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions

Cited by 32 publications

References 22 publications

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription

Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome

Characterization of FFPE-induced bacterial DNA damage and development of a repair method

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