RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity.

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability.

Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms.

DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids.

Break-induced RNA-DNA hybrids (BIRDHs) in homologous recombination: friend or foe?

Double-strand breaks (DSBs) are the most harmful DNA lesions, with a strong impact on cell proliferation and genome integrity. Depending on cell cycle stage, DSBs are preferentially repaired by non-homologous end joining or homologous recombination (HR). In recent years, numerous reports have revealed that DSBs enhance DNA-RNA hybrid formation around the break site.

Non-dipping blood pressure pattern in pediatricians during on-duty.

Introduction: People with a reduced nighttime dip in blood pressure have an increased cardiovascular risk. Our objective was to describe the different patterns in blood pressure (BP) among pediatricians who work in long on-duty shifts in relation with sex, medical rank and sleeping time.

Methods: Descriptive, cross-sectional, two-center study.

A role for the Saccharomyces cerevisiae Rtt109 histone acetyltransferase in R-loop homeostasis and associated genome instability.

The stability of the genome is occasionally challenged by the formation of DNA-RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA-RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability.

Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae.

R-loops are transcriptional by-products formed by a hybrid of the nascent RNA molecule with its DNA template and the displaced nontemplate DNA strand. The single stranded nature of the displaced nontemplate strand makes it vulnerable to attack. This property is used in nature to cause directed mutagenesis and breaks by the action of the activation-induced cytosine deaminase (AID) enzyme and can thus be exploited to detect the presence of R-loops even when they form at low frequencies by overexpressing this enzyme in vivo or by in vitro treatment with the bisulfite anion, which further allows nucleotide resolution.

VID22 counteracts G-quadruplex-induced genome instability.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair.

Analysis of repair of replication-born double-strand breaks by sister chromatid recombination in yeast.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem.

A CDK-regulated chromatin segregase promoting chromosome replication.

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood.

Heterogeneity of DNA damage incidence and repair in different chromatin contexts.

It has been long known that some regions of the genome are more susceptible to damage and mutagenicity than others. Recent advances have determined a critical role of chromatin both in the incidence of damage and in its repair. Thus, chromatin arises as a guardian of the stability of the genome, which is altered in cancer cells.

A new interaction between BRCA2 and DDX5 promotes the repair of DNA breaks at transcribed chromatin.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2-DDX5 interaction is impaired in cells expressing the missense variant found in breast cancer patients.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood.

Origin matters: spontaneous DNA-RNA hybrids do not form in trans as a source of genome instability.

Multiple exogenous and endogenous genotoxic agents threaten the integrity of the genome, but one major source of spontaneous DNA damage is the formation of unscheduled DNA-RNA hybrids. These can be genetically detected by their ability to induce recombination. The origin of spontaneous hybrids has been mainly attributed to the nascent RNA formed co-transcriptionally in cis invading its own DNA template.

Harmful DNA:RNA hybrids are formed and in a Rad51-independent manner.

DNA:RNA hybrids constitute a well-known source of recombinogenic DNA damage. The current literature is in agreement with DNA:RNA hybrids being produced co-transcriptionally by the invasion of the nascent RNA molecule produced in cis with its DNA template. However, it has also been suggested that recombinogenic DNA:RNA hybrids could be facilitated by the invasion of RNA molecules produced in trans in a Rad51-mediated reaction.

Histone H3E73Q and H4E53A mutations cause recombinogenic DNA damage.

The stability and function of eukaryotic genomes is closely linked to histones and to chromatin structure. The state of the chromatin not only affects the probability of DNA to undergo damage but also DNA repair. DNA damage can result in genetic alterations and subsequent development of cancer and other genetic diseases.

Histone deacetylases facilitate the accurate repair of broken forks.

We have recently uncovered that loss of the yeast histone deacetylases Rpd3 (Reduced Potassium Dependency 3) and Hda1 (Histone DeAcetylase 3) affects the cohesion between sister chromatids thus impairing repair of DNA damage at replication forks and enhancing genetic instability. Here we discuss the possible implications of our findings given that histone deacetylases are a promising chemotherapeutic target often used in combination with DNA damaging agents.

Spontaneous DNA-RNA hybrids: differential impacts throughout the cell cycle.

A large body of research supports that transcription plays a major role among the many sources of replicative stress contributing to genome instability. It is therefore not surprising that the DNA damage response has a role in the prevention of transcription-induced threatening events such as the formation of DNA-RNA hybrids, as we have recently found through an siRNA screening. Three major DDR pathways were defined to participate in the protection against DNA-RNA hybrids: ATM/CHK2, ATR/CHK1 and Postreplication Repair (PRR).

Rpd3L and Hda1 histone deacetylases facilitate repair of broken forks by promoting sister chromatid cohesion.

Genome stability involves accurate replication and DNA repair. Broken replication forks, such as those encountering a nick, lead to double strand breaks (DSBs), which are preferentially repaired by sister-chromatid recombination (SCR). To decipher the role of chromatin in eukaryotic DSB repair, here we analyze a collection of yeast chromatin-modifying mutants using a previously developed system for the molecular analysis of repair of replication-born DSBs by SCR based on a mini-HO site.

The DNA damage response acts as a safeguard against harmful DNA-RNA hybrids of different origins.

Despite playing physiological roles in specific situations, DNA-RNA hybrids threat genome integrity. To investigate how cells do counteract spontaneous DNA-RNA hybrids, here we screen an siRNA library covering 240 human DNA damage response (DDR) genes and select siRNAs causing DNA-RNA hybrid accumulation and a significant increase in hybrid-dependent DNA breakage. We identify post-replicative repair and DNA damage checkpoint factors, including those of the ATM/CHK2 and ATR/CHK1 pathways.

Transcription-mediated replication hindrance: a major driver of genome instability.

Genome replication involves dealing with obstacles that can result from DNA damage but also from chromatin alterations, topological stress, tightly bound proteins or non-B DNA structures such as R loops. Experimental evidence reveals that an engaged transcription machinery at the DNA can either enhance such obstacles or be an obstacle itself. Thus, transcription can become a potentially hazardous process promoting localized replication fork hindrance and stress, which would ultimately cause genome instability, a hallmark of cancer cells.