Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes.

Transcription hinders replication fork progression and stability. The ATR checkpoint and specialized DNA helicases assist DNA synthesis across transcription units to protect genome integrity. Combining genomic and genetic approaches together with the analysis of replication intermediates, we searched for factors coordinating replication with transcription. We show that the Sen1/Senataxin DNA/RNA helicase associates with forks, promoting their progression across RNA polymerase II (RNAPII)-transcribed genes. sen1 mutants accumulate aberrant DNA structures and DNA-RNA hybrids while forks clash head-on with RNAPII transcription units. These replication defects correlate with hyperrecombination and checkpoint activation in sen1 mutants. The Sen1 function at the forks is separable from its role in RNA processing. Our data, besides unmasking a key role for Senataxin in coordinating replication with transcription, provide a framework for understanding the pathological mechanisms caused by Senataxin deficiencies and leading to the severe neurodegenerative diseases ataxia with oculomotor apraxia type 2 and amyotrophic lateral sclerosis 4.

PP2A Controls Genome Integrity by Integrating Nutrient-Sensing and Metabolic Pathways with the DNA Damage Response.

Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.

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.

Sch9S6K controls DNA repair and DNA damage response efficiency in aging cells.

Survival from UV-induced DNA lesions relies on nucleotide excision repair (NER) and the Mec1ATR DNA damage response (DDR). We study DDR and NER in aging cells and find that old cells struggle to repair DNA and activate Mec1ATR. We employ pharmacological and genetic approaches to rescue DDR and NER during aging. Conditions activating Snf1AMPK rescue DDR functionality, but not NER, while inhibition of the TORC1-Sch9S6K axis restores NER and enhances DDR by tuning PP2A activity, specifically in aging cells. Age-related repair deficiency depends on Snf1AMPK-mediated phosphorylation of Sch9S6K on Ser160 and Ser163. PP2A activity in old cells is detrimental for DDR and influences NER by modulating Snf1AMPK and Sch9S6K. Hence, the DDR and repair pathways in aging cells are influenced by the metabolic tuning of opposing AMPK and TORC1 networks and by PP2A activity. Specific Sch9S6K phospho-isoforms control DDR and NER efficiency, specifically during aging.

Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing.

The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.

Mechanisms controlling the integrity of replicating chromosomes in budding yeast.

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.

A lethal combination for cancer cells: synthetic lethality screenings for drug discovery.

In recent years, cancer drug discovery has faced the challenging task of integrating the huge amount of information coming from the genomic studies with the need of developing highly selective target-based strategies within the context of tumour cells that experience massive genome instability. The combination between genetic and genomic technologies has been extremely useful and has contributed to efficiently transfer certain approaches typical of basic science to drug discover projects. An example comes from the synthetic lethal approaches, very powerful procedures that employ the rational used by geneticists working on model organisms. Applying the synthetic lethality (SL) screenings to anticancer therapy allows exploiting the typical features of tumour cells, such as genome instability, without changing them, as opposed to the conventional anticancer strategies that aim at counteracting the oncogenic signalling pathways. Recent and very encouraging clinical studies clearly show that certain promising anticancer compounds work through a synthetic lethal mechanism by targeting pathways that are specifically essential for the viability of cancer cells but not of normal cells. Herein we describe the rationale of the synthetic lethality approaches and the potential applications for anticancer therapy.

Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells.

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.

Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase.

S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. We have studied the RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template in sgs1 cells. We show that, in sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein, is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. We suggest that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. Our findings might contribute to explaining some of the recombination defects of BLM cells.