Elongating RNA polymerase II and RNA:DNA hybrids hinder fork progression and gene expression at sites of head-on replication-transcription collisions.

Uncoordinated clashes between replication forks and transcription cause replication stress and genome instability, which are hallmarks of cancer and neurodegeneration. Here, we investigate the outcomes of head-on replication-transcription collisions, using as a model system budding yeast mutants for the helicase Sen1, the ortholog of human Senataxin. We found that RNA Polymerase II accumulates together with RNA:DNA hybrids at sites of head-on collisions. The replication fork and RNA Polymerase II are both arrested during the clash, leading to DNA damage and, in the long run, the inhibition of gene expression. The inactivation of RNA Polymerase II elongation factors, such as the HMG-like protein Spt2 and the DISF and PAF complexes, but not alterations in chromatin structure, allows replication fork progression through transcribed regions. Attenuation of RNA Polymerase II elongation rescues RNA:DNA hybrid accumulation and DNA damage sensitivity caused by the absence of Sen1, but not of RNase H proteins, suggesting that such enzymes counteract toxic RNA:DNA hybrids at different stages of the cell cycle with Sen1 mainly acting in replication. We suggest that the main obstacle to replication fork progression is the elongating RNA Polymerase II engaged in an R-loop, rather than RNA:DNA hybrids per se or hybrid-associated chromatin modifications.

Bi-allelic TARS Mutations Are Associated with Brittle Hair Phenotype.

Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEß), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.

Dormant origins and fork protection mechanisms rescue sister forks arrested by transcription.

The yeast RNA/DNA helicase Sen1, Senataxin in human, preserves the integrity of replication forks encountering transcription by removing RNA-DNA hybrids. Here we show that, in sen1 mutants, when a replication fork clashes head-on with transcription is arrested and, as a consequence, the progression of the sister fork moving in the opposite direction within the same replicon is also impaired. Therefore, sister forks remain coupled when one of the two forks is arrested by transcription, a fate different from that experienced by forks encountering Double Strand Breaks. We also show that dormant origins of replication are activated to ensure DNA synthesis in the proximity to the forks arrested by transcription. Dormant origin firing is not inhibited by the replication checkpoint, rather dormant origins are fired if they cannot be timely inactivated by passive replication. In sen1 mutants, the Mre11 and Mrc1-Ctf4 complexes protect the forks arrested by transcription from processing mediated by the Exo1 nuclease. Thus, a harmless head-on replication-transcription clash resolution requires the fine-tuning of origin firing and coordination among Sen1, Exo1, Mre11 and Mrc1-Ctf4 complexes.

2D Gel Electrophoresis to Detect DNA Replication and Recombination Intermediates in Budding Yeast.

The two-dimensional agarose gel electrophoresis (2D gel) is a powerful method used to detect and analyze rare DNA replication and recombination intermediates within a genomic DNA preparation. The 2D gel method has been extensively applied to the budding yeast Saccharomyces cerevisiae due to its small and well-characterized genome to analyze replication fork dynamics at single DNA loci under both physiological and pathological conditions. Here we describe procedures to extract genomic DNA from in vivo UV-psoralen cross-linked yeast cells, to separate branched DNA replication and recombination intermediates by neutral-neutral 2D gel method and to visualize 2D gel structures by Southern Blot.

The dark side of RNA:DNA hybrids.

RNA:DNA hybrids form when nascent transcripts anneal to the DNA template strand or any homologous DNA region. Co-transcriptional RNA:DNA hybrids, organized in R-loop structures together with the displaced non-transcribed strand, assist gene expression, DNA repair and other physiological cellular functions. A dark side of the matter is that RNA:DNA hybrids are also a cause of DNA damage and human diseases. In this review, we summarize recent advances in the understanding of the mechanisms by which the impairment of hybrid turnover promotes DNA damage and genome instability via the interference with DNA replication and DNA double-strand break repair. We also discuss how hybrids could contribute to cancer, neurodegeneration and susceptibility to viral infections, focusing on dysfunctions associated with the anti-R-loop helicase Senataxin.

Senataxin Ortholog Sen1 Limits DNA:RNA Hybrid Accumulation at DNA Double-Strand Breaks to Control End Resection and Repair Fidelity.

An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.

Replication and transcription on a collision course: eukaryotic regulation mechanisms and implications for DNA stability.

DNA replication and transcription are vital cellular processes during which the genetic information is copied into complementary DNA and RNA molecules. Highly complex machineries required for DNA and RNA synthesis compete for the same DNA template, therefore being on a collision course. Unscheduled replication-transcription clashes alter the gene transcription program and generate replication stress, reducing fork speed. Molecular pathways and mechanisms that minimize the conflict between replication and transcription have been extensively characterized in prokaryotic cells and recently identified also in eukaryotes. A pathological outcome of replication-transcription collisions is the formation of stable RNA:DNA hybrids in molecular structures called R-loops. Growing evidence suggests that R-loop accumulation promotes both genetic and epigenetic instability, thus severely affecting genome functionality. In the present review, we summarize the current knowledge related to replication and transcription conflicts in eukaryotes, their consequences on genome stability and the pathways involved in their resolution. These findings are relevant to clarify the molecular basis of cancer and neurodegenerative diseases.