Transcription as a Threat to Genome Integrity

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant

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. Understanding the causes behind transcription–replication conflicts as well as how the cell resolves them to sustain genome integrity is the aim of this review

Gene gating at nuclear pores prevents the formation of R loops

Transcription is an important source of genetic variability. A large amount of transcription-associated genome variation arises from the unscheduled formation of R loops. We have recently found that physical proximity of chromatin to nuclear pores prevents the formation of pathological R loops during transcription. Our study opens new perspectives to understand genome stability as a function of nuclear location

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. We confirm the candidates through FLP-based systems based on a mutated version of the FLP flipase that causes nicks on either the leading or lagging DNA strands. We demonstrate that Rpd3L and Hda1 histone deacetylase (HDAC) complexes contribute to the repair of replication-born DSBs by facilitating cohesin loading, with no effect on other types of homology-dependent repair, thus preventing genome instability. We conclude that histone deacetylation favors general sister chromatid cohesion as a necessary step in SCR

Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability

R loops are nucleic acid structures comprising an DNA-RNA hybrid and a displaced single-stranded DNA. These structures may occur transiently during transcription, playing essential biological functions. However, persistent R loops may become pathological as they are important drivers of genome instability and have been associated with human diseases. The mitochondrial degradosome is a functionally conserved complex from bacteria to human mitochondria. It is composed of the ATP-dependent RNA and DNA helicase SUV3 and the PNPase ribonuclease, playing a central role in mitochondrial RNA surveillance and degradation. Here we describe a new role for the mitochondrial degradosome in preventing the accumulation of pathological R loops in the mitochondrial DNA, in addition to preventing dsRNA accumulation. Our data indicate that, similar to the molecular mechanisms acting in the nucleus, RNA surveillance mechanisms in the mitochondria are crucial to maintain its genome integrity by counteracting pathological R-loop accumulation.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-

Detection of DNA-RNA hybrids in vivo

DNA-RNA hybrids form naturally during essential cellular functions such as transcription and replication. However, they may be an important source of genome instability, a hallmark of cancer and genetic diseases. Detection of DNA-RNA hybrids in cells is becoming crucial to understand an increasing number of molecular biology processes in genome dynamics and function and to identify new factors and mechanisms responsible for disease in biomedical research. Here, we describe two different procedures for the reliable detection of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae and in human cells: DNA-RNA Immunoprecipitation (DRIP) and Immunofluorescence

Detection of DNA Double-Strand Breaks byγ-H2AX Immunodetection

DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of geneticinstability as they can lead to mutations, genome rearrangements, or loss of genetic material when notproperly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBswith the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated inserine 139 in mammals (S129 in yeast), is namedγ-H2AX. Detection of DSBs is of high relevance inresearch on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association ofDSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels ofγ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter sometechniques to detectγ-H2AX are described

The role of chromatin at transcription-replication conflicts as a genome safeguard

DNA replication ensures the correct copying of the genome and the faithful transfer of the genetic information to the offspring. However, obstacles to replication fork (RF) progression cause RF stalling and compromise efficient genome duplication. Since replication uses the same DNA template as transcription, both transcription and replication must be coordinated to prevent Transcription-Replication Conflicts (TRCs) that could stall RF progression. Several factors contribute to limit the occurrence of such conflicts and their harmful impact on genome integrity. Increasing evidence indicates that chromatin homeostasis plays a key role in the cellular response to TRCs as well as in the preservation of genome integrity. Indeed, chromatin regulating enzymes are frequently mutated in cancer cells, a common characteristic of which is genome instability. Therefore, understanding the role of chromatin in TRC occurrence and resolution may help identify the molecular mechanism by which chromatin protects genome integrity, and the causes and physiological relevance of the high mutation rates of chromatin regulating factors in cancer. Here we review the current knowledge in the field, as well as the perspectives and future applications

The SWI/SNF complex, transcription-replication conflicts and cancer: a connection with high therapeutic potential

Genome instability is a hallmark of cancer. ATP-dependent chromatin remodelers are frequently altered in cancer. We have recently reported that the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protects the genome by limiting R-loop-mediated genome instability, mainly that caused by transcription-replication conflicts. Here we discuss the significance and biomedical applications of this finding

DNA-RNA hybrids: The risks of DNA breakage during transcription

Although R loops can occur at different genomic locations, the factors that determine their formation and frequency remain unclear. Emerging evidence indicates that DNA breaks stimulate DNA-RNA hybrid formation. Here, we discuss the possibility that formation of hybrids may be an inevitable risk of DNA breaks that occur within actively transcribed regions. While such hybrids must be removed to permit repair, their potential role as repair intermediates remains to be established