DNA G-quadruplexes: Structural and functional insights.

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Timeless, an integral component of the replication fork protection complex, stabilizes the replication fork in response to DNA damage. Timeless is also involved with the maintenance of genomic integrity through its interaction with downstream targets that aid in checkpoint regulation, DNA repair, and DNA replication. Recent studies have shown that Timeless interacts with PARP1 to promote DNA repair via homologous recombination. Timeless also interacts with DDX11 to facilitate replication at hard-to-replicate regions of the genome. Studies have suggested the role of Timeless in cancer progression; Timeless is frequently overexpressed in various types of cancers, and this high expression is associated with poor patient prognosis and increased metastasis. Furthermore, Timeless confers resistance to chemotherapeutic drugs such as cisplatin and serves as a marker for tamoxifen resistance. However, the mechanisms by which Timeless regulates DNA repair and carcinogenesis are not well understood. In this study, we aimed to understand the role of Timeless in genome maintenance and cancer phenotypes. We performed mutational analysis of Timeless to determine the role of Timeless-PARP1 interaction in DNA repair and Timeless-DDX11 interaction in DNA replication. We found that Timeless depletion leads to growth inhibition and a significant increase in senescence-associated phenotypes. Considering that senescence is generally regarded as a tumor-suppressive mechanism, our findings suggest that Timeless promote survival of cancer cells. Future studies would include assessing the effects of Timeless mutations in DNA replication, repair, and cancer phenotypes. We expect that these studies will serve as a stepping-stone to develop effective strategies for cancer therapy.

G-quadruplexes (G4s) are secondary structures abundant in DNA that may play regulatory roles in cells. Despite the ubiquity of the putative G-quadruplex-forming sequences (PQS) in the human genome, only a small fraction forms G4 structures in cells. Folded G4, histone methylation and chromatin accessibility are all parts of the complex cis regulatory landscape. We propose an approach for prediction ofG4 formation in cells that incorporates epigenetic and chromatin accessibility data. The novel approach termed epiG4NN efficiently predicts cell-specific G4 formation in live cells based on a local epigenomic snapshot. Our results confirm the close relationship between H3K4me3 histone methylation, chromatin accessibility and G4 structure formation. Trained on A549 cell data, epiG4NN was then able to predict G4 formation in HEK293T and K562 cell lines. We observe the dependency of model performance with different epigenetic features on the underlying experimental condition of G4 detection. We expect that this approach will contribute to the systematic understanding of correlations between structural and epigenomic feature landscape.

G-quadruplex (G4) DNA structures are involved in many important biological processes and can be linked to several human diseases. Drug-like low molecular weight compounds that target G4 structures are therefore interesting not only for their potential therapeutic properties but also for their potential use as chemical research tools. We report here on the development of methods to synthesize spiropyrans using a condensation-cyclisation reaction of quaternary salts of α-methyl quinoline or phenanthridine with salicylaldehydes. Evaluation of the synthesized phenanthridine spiropyrans' interactions with G4 DNA was performed with a Thioflavin T displacement assay, circular dichroism, Taq DNA polymerase stop assay, and NMR. This revealed that the substitution pattern on the phenanthridine spiropyrans was very important for their ability to bind and stabilize G4 structures. Some of the synthesized low molecular weight spirocyclic compounds efficiently stabilized G4 structures without inducing structural changes by binding the first G-tetrad in the G4 structure.

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.

Detecting G4 unwinding.

G-quadruplex (G4) structures are important in both biological and medical fields. A unique G4 type containing G vacancies allowing stabilization by guanine or guanine derivatives is widely used in the treatment of diseases, such as cancer. However, the identification of ligands with suitable affinities and specificities for G4 structures remains challenging. This study investigated the assisting effect of guanine derivatives in the formation of G-vacancy G4 structures. Focusing on the EAD2 sequence, we designed EAD2 sequences with one or two missing G bases. Circular dichroism (CD) assays revealed negligible impact of guanine and its derivatives on intact EAD2 structures but notable alterations in single-G-base-deficient EAD2 sequences. The formation of G4 structures in the EAD2 sequence lacking two G bases was difficult, even with the assistance of guanine derivatives. Moreover, experimental results demonstrated that the guanine derivatives M2 had better auxiliary effects on base-deficient sequences than other compounds, which may be due to the enhanced pairing capability of G4 caused by M2. Lastly, the experiment demonstrates that the hemin/G4 DNAzyme-catalyzed ABTS confirmed the auxiliary role of the guanine derivatives in the formation of G4 in G base-deficient sequences. Overall, the findings indicated the significant impact of G base deficiency on G4 assembly, with the guanine derivative M2 effectively aiding stable G4 formation in G base-deficient sequences.

Cigarette smoke (CS) is a major risk factor for many respiratory diseases, including lung cancer, contributing to genomic instability, chronic inflammation, and impaired immune responses. Macrophages are among the most affected cell types, and CS-induced polarization has been increasingly associated with tumor promotion and reduced anti-tumor activity. G quadruplexes (G4) are non-canonical structures of RNA and DNA, enriched in G-rich sequences (such as telomeres or gene promoters), influencing several layers of gene expression regulation and DNA stability. More recently, genome-wide approaches have enabled high-resolution mapping of G4 structures in human cells, confirming the formation and functionality of G4 in multiple biological contexts. G4 structures can be bound and stabilized by small molecules such as RHPS4, with a consequent impairment of cell proliferation in cancer cells and downregulation of several pro-oncogenic factors. However, no studies have previously addressed whether CS exposure affects G4 formation or stability. Here, we found that CS exposure destabilizes G4 structure formation in cultured THP-1 monocytes differentiated into macrophages, antagonizing the effect of the G4 ligand RHPS4. In this context, CS exposure strongly induces the activation of IL-1β and TNF-α, both containing a G4 putative forming region in their promoters, suggesting that CS-linked cytokine modulation could involve a G4-dependent mechanism. Moreover, in proliferating THP-1, CS antagonizes the anti-proliferative effect of RHPS4 and has an opposite effect on the expression of G4 containing pro-oncogenic genes (like myc and bcl2). Overall, these findings showed for the first time a relationship between CS exposure and G4 structures, representing a new unexplored regulatory mechanism underlying smoking-related conditions and carcinogenesis.

Alternaria alternata is a common airborne fungus that can be found in both indoor and outdoor environments. It is a major allergen linked to respiratory diseases such as allergic rhinitis and asthma. It may also infect the skin, nails, or eyes. In addition, uncharacterized or hypothetical proteins are present but have not yet been identified or associated with specific protein sequences. Domains of undetermined activity refer to molecules with known, experimentally determined activity. Yet, the exact role or structural features of these proteins have not been identified. Many proteins encoded in A. alternata genome remain uncharacterized, including the hypothetical protein XP_018378625.1 containing the SRC1lrK2f domain, whose function is unknown. This research employed a comprehensive in-silico approach to predict its physicochemical features, structural properties, subcellular localization, and protein-protein interactions and 3D structure. The protein was found to be hydrophilic, with an instability index of 46.62 and isoelectric point value of 6.41 which indicates acidic nature. The in-silico analysis indicated that the protein is soluble, with its secondary structure mainly composed of a random coil suggesting structural flexibility. The analysis showed that the protein contains a cytoplasmic domain. Moreover, the protein-protein interactions were examined using STRING software, which showed that the SRC1lrK2f protein has a strong interaction with exodeoxyribonuclease 1, implying a possible role in DNA repair or genome maintenance. These findings provide the first functional insights into the SRC1lrK2f domain in A. alternata and may support the future development of pharmaceutical strategies for managing Alternaria-induced allergic and infectious diseases by advancing our understanding of the biological role of the SRC1lrK2f domain.

Hepatitis C virus (HCV) is the major cause of chronic liver disease and hepatocellular carcinoma. It is currently estimated that over 71 million people in the world are chronically infected with HCV. The current HCV drugs are extremely expensive, and thus they are not available for the majority of patients infected with the virus. Moreover, due to high rate of mutation of the HCV genome, it is likely that the virus develops resistance to these drugs. Therefore, it is important to develop a cost‐effective anti‐viral compound that targets conserved regions within the HCV genome and prevent replication of various HCV genotypes and subtypes.The HCV genome encodes for ten different viral proteins, one of which is Non‐structural protein 3 (NS3). NS3 has both protease and helicase domain. The helicase domain of NS3 is essential for viral replication. However, its mechanism of action is not well understood. Previous works have shown the presence of conserved guanine‐rich consensus sequences within the HCV genome. These guanine rich sequences form G‐quadruplex (G4) secondary structures through Hoogesteen hydrogen bonding. It has been reported that both replication and translation of HCV genome could be inhibited by the formation of G4 structures. However, it is still unknown how these HCV G4RNA structures are regulated. We hypothesized that the Helicase domain of NS3 facilitates HCV replication by unfolding conserved G4 structures within the HCV genome.To investigate whether NS3 helicase resolves HCV G4RNA structures, we purchased synthetic HCV guanine rich RNA sequences and performed circular dichroism spectroscopy to confirm G4 formation. In addition, fluorescence anisotropy NS3 binding and trap unfolding assays were conducted. We showed that NS3 binds and resolves HCV G4RNA structure in an ATP dependent manner. Understanding the mechanism by which NS3 resolves HCV G4RNA might allow us to find processes and factors that could be targeted to prevent HCV replication in a host cell.Support or Funding InformationThis work is supported by MIRA grant R35GM122601This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Triple negative breast cancers (TNBC) represent a heterogeneous group of patients where pathway targeted interventions are lacking. We have recently identified new subgroups of TNBC tumours based on patterns of genomic instability, suggesting that deficiencies in DNA repair or genome maintenance could be exploited in specific subgroups of TNBC patients. We recently discovered synthetic lethal activity for small molecules that bind and stabilise G-quadruplex (G4) structures in the human genome. Stabilized G4 structures are considered "at risk" during DNA replication, leading to single strand and double strand breaks in the absence of DNA repair. This has specific application in cancers with DNA repair deficiency. We have shown that loss of homologous recombination repair capacity in breast cancers is associated with ∼ 1 log order increase in sensitivity to G4 binders. Moreover this activity has been observed in platinum pre-treated tumours, and in PARP insensitive tumours. Recently through CRISPR screening approaches we have defined additional DNA repair lesions that confer synthetic lethality to G4 binders. This mechanism of G4 stabilisation is now being tested in a phase 1/2 clinical trial of CX5461 in breast cancer patients. Citation Format: Aparicio S. Targeting DNA repair deficiency in triple negative breast cancers (TNBC) with G-quadruplex stabilisers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr MS1-1.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

G-Quadruplex (G4) DNA is a DNA structure that can occur in G-rich DNA sequences. These structures, which are compact and diverse, have been implicated in a wide range of processes including telomere maintenance, transcriptional control, and DNA replication and repair. In order to target specific G4 structures, and thus selectively affect these functions, improved small-molecule ligands are required. We have carried out a docking study to explore the structural origins of ligand selectivity for different topological families of G4 structures. Using Autodock Vina, we have analyzed the G4 binding affinity, mode, and selectivity of previous reported and newly designed bis(quinolin-3-ylmethylene)-based ligands towards hybrid and parallel stranded G-quadruplex structures. Based on this analysis, we have embarked on the synthesis of one such novel bis(quinolin-3-ylmethylene)-based ligand, which is predicted to be more selective for parallel-stranded G4 structures. In addition, this ligand is predicted to be more readily taken up by cells compared to the parent bis(quinolin-3-ylmethylene)-based ligands.

ABSTRACTMany factors may affect the chances for successful pregnancy, especially at a later age. Fertility evaluations including genetic analysis are recommended to couples that have not achieved pregnancy within 6–12 months of unprotected intercourse. This review discusses some of the common polymorphisms in genes coding for proteins functioning in DNA damage identification and repair and maintenance of genomic integrity that may affect the chances of success in natural conception as well as in assisted reproduction (AR). Common polymorphisms in genes coding for proteins functioning in DNA damage identification and repair and maintenance of genomic integrity may affect the chances of success in assisted reproduction as well as in natural conception. The effects of carriership of different alleles of key genes of DNA repair may have differential effects in men and women and at different ages, suggesting complex interactions with the mechanisms controlling cell and tissue aging and programmed cell death. Future studies in the field are needed in order to elucidate the genotype–phenotype relationships and to translate the knowledge about individual repair capacity and maintenance of genomic integrity to potential clinical applications.Abbreviations: aCGH: microarray-based comparative genomic hybridization; AR: assisted reproduction; ATM: ataxia-telangiectasia mutated; ATP: adenosine triphosphate; BER: base excision repair; BFE: basic fertility evaluation; DMSO: dimethyl sulfoxide; FSH: follicle-stimulating hormone; GNRHR: gonadotropin-releasing hormone receptor; HMG: high-mobility group; ICSI: intracytoplasmic sperm injection; IUI: intrauterine insemination; IVF: in vitro fertilization; LH: luteinizing hormone; LIF: leukaemia inhibitory factor; MTR: methionine synthase; MTRR: methionine synthase reductase; NGS: next-generation sequencing; NER: nucleotide excision repair; NHEJ: non-homologous end joining; PAH: polycyclic aromatic hydrocarbons; PCOS: polycystic ovarian syndrome; R: restriction point; ROS: reactive oxygen species; VEGF: vascular endothelial growth factor

CX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.