Spotlight on G-Quadruplexes: From Structure and Modulation to Physiological and Pathological Roles.

G-quadruplexes or G4s are non-canonical secondary structures of nucleic acids characterized by guanines arranged in stacked tetraplex arrays. Decades of research into these peculiar assemblies of DNA and RNA, fueled by the development and optimization of a vast array of techniques and assays, has resulted in a large amount of information regarding their structure, stability, localization, and biological significance in native systems. A plethora of articles have reported the roles of G-quadruplexes in multiple pathways across several species, ranging from gene expression regulation to RNA biogenesis and trafficking, DNA replication, and genome maintenance. Crucially, a large amount of experimental evidence has highlighted the roles of G-quadruplexes in cancer biology and other pathologies, pointing at these structurally unique guanine assemblies as amenable drug targets. Given the rapid expansion of this field of research, this review aims at summarizing all the relevant aspects of G-quadruplex biology by combining and discussing results from seminal works as well as more recent and cutting-edge experimental evidence. Additionally, the most common methodologies used to study G4s are presented to aid the reader in critically interpreting and integrating experimental data.

PARylation of BRCA1 limits DNA break resection through BRCA2 and EXO1.

The nucleolytic processing (resection) of a DNA double-strand break (DSB) is a critical step to repair the lesion by homologous recombination (HR). PARylation, which is the attachment of poly(ADP-ribose) (PAR) units to specific targets by PAR polymerases (PARPs), regulates many steps of HR, including resection. Here, we show that preventing PARylation of the oncosuppressor BRCA1 induces hyper-resection of DSBs through BRCA2 and the EXO1 nuclease. Upon expression of the unPARylatable variant of BRCA1, we observe a reduced 53BP1-RIF1 barrier for resection accompanied by an increase in the recruitment of the RAD51 recombinase. Similar results are observed when cells are treated with the clinically approved PARP inhibitor olaparib. We propose that PARylation of BRCA1 is important to limit the formation of excessively extended DNA filaments, thereby reducing illegitimate chromosome rearrangements. Our results shed light on molecular aspects of HR and on the mechanisms of PARP inhibitor treatment.

Inhibition of DNA Repair in Cancer Therapy: Toward a Multi-Target Approach.

Alterations in DNA repair pathways are one of the main drivers of cancer insurgence. Nevertheless, cancer cells are more susceptible to DNA damage than normal cells and they rely on specific functional repair pathways to survive. Thanks to advances in genome sequencing, we now have a better idea of which genes are mutated in specific cancers and this prompted the development of inhibitors targeting DNA repair players involved in pathways essential for cancer cells survival. Currently, the pivotal concept is that combining the inhibition of mechanisms on which cancer cells viability depends is the most promising way to treat tumorigenesis. Numerous inhibitors have been developed and for many of them, efficacy has been demonstrated either alone or in combination with chemo or radiotherapy. In this review, we will analyze the principal pathways involved in cell cycle checkpoint and DNA repair focusing on how their alterations could predispose to cancer, then we will explore the inhibitors developed or in development specifically targeting different proteins involved in each pathway, underscoring the rationale behind their usage and how their combination and/or exploitation as adjuvants to classic therapies could help in patients clinical outcome.

Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases.

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.

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.

Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR.

Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the kinetic of formation and 5'-end nucleolytic processing of Cas9-induced DSB in different human cells lines. Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNA-PKcs and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome manipulation.

TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9.

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1-TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair.

Reduced kinase activity of polo kinase Cdc5 affects chromosome stability and DNA damage response in S. cerevisiae.

Polo-like kinases (PLKs) control several aspects of eukaryotic cell division and DNA damage response. Remarkably, PLKs are overexpressed in several types of cancer, being therefore a marker of bad prognosis. As such, specific PLK kinase activity inhibitors are already used in clinical trials and the regulation of PLK activation is a relevant topic of cancer research. Phosphorylation of threonine residues in the T-loop of the kinase domain is pivotal for PLKs activation. Here, we show that T238A substitution in the T-loop reduces the kinase activity of Cdc5, the only PLK in Saccharomyces cerevisiae, with minor effect on cell growth in unperturbed conditions. However, the cdc5-T238A cells have increased rate of chromosome loss and gross chromosomal rearrangements, indicating altered genome stability. Moreover, the T238A mutation affects timely localization of Cdc5 to the spindle pole bodies and blocks cell cycle restart after one irreparable double-strand break. In cells responding to alkylating agent metylmethane sulfonate (MMS), the cdc5-T238A mutation reduces the phosphorylation of Mus81-Mms4 resolvase and exacerbates the MMS sensitivity of sgs1Δ cells that accumulate Holliday junctions. Of importance, the previously described checkpoint adaptation defective allele, cdc5-ad does not show reduced kinase activity, defective Mms4 phosphorylation and genetic interaction with sgs1Δ. Our data define the importance of regulating Cdc5 activity through T-loop phosphorylation to preserve genome integrity and respond to DNA damage.

To trim or not to trim: progression and control of DSB end resection.

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5'-3' nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.

The RSC chromatin-remodeling complex influences mitotic exit and adaptation to the spindle assembly checkpoint by controlling the Cdc14 phosphatase.

Upon prolonged activation of the spindle assembly checkpoint, cells escape from mitosis through a mechanism called adaptation or mitotic slippage, which is thought to underlie the resistance of cancer cells to antimitotic drugs. We show that, in budding yeast, this mechanism depends on known essential and nonessential regulators of mitotic exit, such as the Cdc14 early anaphase release (FEAR) pathway for the release of the Cdc14 phosphatase from the nucleolus in early anaphase. Moreover, the RSC (remodel the structure of chromatin) chromatin-remodeling complex bound to its accessory subunit Rsc2 is involved in this process as a novel component of the FEAR pathway. We show that Rsc2 interacts physically with the polo kinase Cdc5 and is required for timely phosphorylation of the Cdc14 inhibitor Net1, which is important to free Cdc14 in the active form. Our data suggest that fine-tuning regulators of mitotic exit have important functions during mitotic progression in cells treated with microtubule poisons and might be promising targets for cancer treatment.

Elevated levels of the polo kinase Cdc5 override the Mec1/ATR checkpoint in budding yeast by acting at different steps of the signaling pathway.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

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.

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1.

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

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.