Rodrigo Bermejo

Topoisomerase I poisoning results in PARP-mediated replication fork reversal

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress–inducing chemotherapeutics.

G-quadruplex-induced instability during leading-strand replication.

G‐quadruplexes are four‐stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G‐quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G‐quadruplex‐prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild‐type cells but is very frequently destabilized upon treatment with the potent Phen‐DC3 G‐quadruplex ligand, or in the absence of the G‐quadruplex‐unwinding Pif1 helicase, only when the G‐rich strand is the template of leading‐strand replication. The orientation‐dependent instability is associated with the formation of Rad51–Rad52‐dependent X‐shaped intermediates during replication detected by two‐dimensional (2D) gels, and relies on the presence of intact G‐quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G‐quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G‐rich telomeres.

The Replication Checkpoint Protects Fork Stability by Releasing Transcribed Genes from Nuclear Pores

Summary Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Senataxin Associates with Replication Forks to Protect Fork Integrity across RNA-Polymerase-II-Transcribed Genes

Summary 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.

Genome‐wide function of THO/TREX in active genes prevents R‐loop‐dependent replication obstacles

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription‐associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)‐chip studies reveal that the Hpr1 component of THO and the Sub2 RNA‐dependent ATPase have genome‐wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5′ → 3′ gradient. This is accompanied by a genome‐wide impact of THO–Sub2 deletions on expression of highly expressed, long and high G+C‐content genes. Importantly, ChIP‐chips reveal an over‐recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome‐wide function for THO–Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription‐mediated replication obstacles, including R‐loops.

Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements.

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions.

Replicon Dynamics, Dormant Origin Firing, and Terminal Fork Integrity after Double-Strand Break Formation

In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres.

Genome-Organizing Factors Top2 and Hmo1 Prevent Chromosome Fragility at Sites of S phase Transcription

Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination‐mediated (error‐free) and translesion synthesis‐mediated (error‐prone) DNA damage tolerance pathways. Crucial for error‐free DNA damage tolerance is template switching, which depends on the formation and resolution of damage‐bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication‐associated lesions into the error‐free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C‐terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication‐associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

ChIP-on-Chip Analysis of DNA Topoisomerases

Here we describe an adapted ChIP-on-chip protocol for the analysis of DNA topoisomerase chromosomal binding in Saccharomyces cerevisiae cells. The ChIP-on-chip technique is based on the immunoprecipitation of crosslinked chromatin (ChIP, chromatin immunoprecipitation), followed by DNA amplification and hybridization to high-density oligonucleotide arrays (Chip). Comparison of the signal intensities of immunoprecipitated and control fractions provides a measurement of the protein-DNA association along entire genomes. ChIP-on-chip analysis of DNA topoisomerase binding to chromosomal DNA opens a window to the understanding of the in vivo contribution of these enzymes to the different DNA transactions taking place concomitantly within the context of the highly organized eukaryotic genome. Chromosomal binding profiles obtained from synchronized cells allow scoring the temporal and spatial restriction of these enzymes at different cell cycle stages. By using this approach, novel aspects of DNA topoisomerase function in chromosome metabolism might be unmasked.