ATP-dependent DNA Helicase Research Articles

Abstract 3113: Development of HTS enzymatic assays for WRN helicase and exonuclease functions

Abstract WRN, a double strand break repair enzyme with ATP-dependent DNA-helicase and 3’- >5’ exonuclease activities, is under intense focus as a therapeutic target because of its synthetic lethality in tumors with microsatellite instability. To accelerate discovery of WRN inhibitors, we developed and validated homogenous, fluorescence-based high throughput assays for WRN helicase and exonuclease enzymatic activities using the Transcreener HTS Assay platform, which relies on highly selective antibodies to detect nucleotides in a homogenous (mix-and-read) format with far red fluorescent readouts. We produced the WRN helicase domain (aa 500-946, N-terminal 6xHis) in BaV-infected insect cells and optimized detection of its dsDNA-dependent ATPase activity using the Transcreener ADP2 Assay in FP, TR-FRET, and FI detection modes. Optimal reaction conditions included 50 μM ATP (approximate Km) and saturating concentration (100 nM) of a 3’ flapped duplex DNA oligomer; these conditions yielded a good signal with 250 pM WRN, a kcat of 300 min−1, and a Z’ of 0.85. An IC50 of 35 nM was determined for the probe inhibitor NSC-617145, and a 1,280 compound pilot screen (Tocris 2.0 Micro) identified multiple hits that were confirmed in dose response experiments. We produced the WRN 3’-5’ exonuclease domain (aa 1-133-6xHis) in E. coli and used the Transcreener dAMP Exonuclease Assay to allow direct, FP-based detection of dAMP monomers released from a 52 bp dsDNA oligomer with a 5’ overhang. With saturating DNA present (1 μM), the assay produced a good signal with 1 nM enzyme; the Z’ value was 0.89. There are no published inhibitors for the WRN exonuclease, but suramin inhibited with an IC50 of approximately 100 nM. These assays allow sensitive, robust, quantitative detection of WRN ATPase and exonuclease activities using an extensively validated HTS platform; they should be valuable tools for screening and optimizing small molecules targeting specific WRN functions. Citation Format: Ha Pham, Mahbbat Ali, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for WRN helicase and exonuclease functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3113.

Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model

Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5′-GCTGGTGG-3′). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model – upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.

Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

Spin-dependent electrified protein interfaces for probing the CISS effect.

Bio-spinterfaces present numerous opportunities to study spintronics across the biomolecules attached to (ferro)magnetic electrodes. While it offers various exciting phenomena to investigate, it is simultaneously challenging to make stable bio-spinterfaces as biomolecules are sensitive to many factors that it encounters during thin-film growth to device fabrication. The chirality-induced spin-selectivity effect is an exciting discovery, demonstrating an understanding that a specific electron's spin (either up or down) passes through a chiral molecule. The present work utilizes Ustilago maydis Rvb2 protein, an ATP-dependent DNA helicase (also known as Reptin), to fabricate bio-spintronic devices to investigate spin-selective electron transport through the protein. Ferromagnetic materials are well-known for exhibiting spin-polarization, which many chiral and biomolecules can mimic. We report herein spin-selective electron transmission through Rvb2 that exhibits 30% spin polarization at a low bias (+0.5V) in a device configuration, Ni/Rvb2 protein/indium tin oxide measured under two different magnetic configurations. Our findings demonstrate that biomolecules can be put in circuit components without any expensive vacuum deposition for the top contact. The present study holds a remarkable potential to advance spin-selective electron transport in other biomolecules, such as proteins and peptides, for biomedical applications.

New insights into the antimicrobial action and protective therapeutic effect of tirapazamine towards Escherichia coli-infected mice

Escherichia coli is an important pathogen responsible for numerous cases of diarrhoea worldwide. The bioreductive agent tirapazamine (TPZ), which was clinically used to treat various types of cancers, has obvious antibacterial activity against E. coli strains. In the present study, we aimed to evaluate the protective therapeutic effects of TPZ in E. coli-infected mice and provide insights into its antimicrobial action mechanism. The MIC and MBC tests, drug sensitivity test, crystal violet assay and proteomic analysis were used to detect the in vitro antibacterial activity of TPZ. The clinical symptoms of infected mice, tissue bacteria load, histopathological features and gut microbiota changes were regarded as indicators to evaluation the efficacy of TPZ in vivo. Interestingly, TPZ-induced the reversal of drug resistance in E. coli by regulating the expression of resistance-related genes, which may have an auxiliary role in the clinical treatment of drug-resistant bacterial infections. More importantly, the proteomics analysis showed that TPZ upregulated 53 proteins and downregulated 47 proteins in E. coli. Among these, the bacterial defence response-related proteins colicin M and colicin B, SOS response-related proteins RecA, UvrABC system protein A, and Holliday junction ATP-dependent DNA helicase RuvB were all significantly upregulated. The quorum sensing-related protein glutamate decarboxylase, ABC transporter-related protein glycerol-3-phosphate transporter polar-binding protein, and ABC transporter polar-binding protein YtfQ were significantly downregulated. The oxidoreductase activity-related proteins pyridine nucleotide-disulfide oxidoreductase, glutaredoxin 2 (Grx2), NAD(+)-dependent aldehyde reductase, and acetaldehyde dehydrogenase, which participate in the elimination of harmful oxygen free radicals in the oxidation-reduction process pathway, were also significantly downregulated. Moreover, TPZ improved the survival rate of infected mice; significantly reduced the bacteria load in the liver, spleen, and colon; and alleviated E. coli-associated pathological damages. The gut microbiota also changed in TPZ-treated mice, and these genera were considerably differentiated: Candidatus Arthromitus, Eubacterium coprostanoligenes group, Prevotellaceae UCG-001, Actinospica, and Bifidobacterium. TPZ may represent an effective and promising lead molecule for the development of antimicrobial agents for the treatment of E. coli infections.

Activity, substrate preferenceand structure of the HsMCM8/9 helicase.

The minichromosomal maintenance proteins, MCM8 and MCM9, are more recent evolutionary additions to the MCM family, only cooccurring in selected higher eukaryotes. Mutations in these genes are directly linked to ovarian insufficiency, infertility, and several cancers. MCM8/9 appears to have ancillary roles in fork progression and recombination of broken replication forks. However, the biochemical activity, specificitiesand structures have not been adequately illustrated, making mechanistic determination difficult. Here, we show that human MCM8/9 (HsMCM8/9) is an ATP dependent DNA helicase that unwinds fork DNA substrates with a 3'-5' polarity. High affinity ssDNA binding occurs in the presence of nucleoside triphosphates, while ATP hydrolysis weakens the interaction with DNA. The cryo-EM structure of theHsMCM8/9 heterohexamer was solved at 4.3 Å revealing a trimer of heterodimer configuration with two types of interfacial AAA+ nucleotide binding sites that become more organized upon binding ADP. Local refinements of the N or C-terminal domains (NTD or CTD) improved the resolution to 3.9 or 4.1 Å, respectively, and shows a large displacement in the CTD. Changes in AAA+ CTD upon nucleotide binding and a large swing between the NTD and CTD likely implies that MCM8/9 utilizes a sequential subunit translocation mechanism for DNA unwinding.

Insights into the Oncogenic, Prognostic, and Immunological Role of BRIP1 in Pan-Cancer: A Comprehensive Data-Mining-Based Study.

BRCA1 interacting helicase 1 (BRIP1), an ATP-dependent DNA helicase which belongs to an Iron-Sulfur (Fe-S) helicase cluster family with a DEAH domain, plays a key role in DNA damage and repair, Fanconi anemia, and development of several cancers including breast and ovarian cancer. However, its role in pan-cancer remains largely unknown. BRIP1 expression data of tumor and normal tissues were downloaded from the Cancer Genome Atlas, Genotype-Tissue Expression, and Human Protein Atlas databases. Correlation between BRIP1 and prognosis, genomic alterations, and copy number variation (CNV) as well as methylation in pan-cancer were further analyzed. Protein-protein interaction (PPI) and gene set enrichment and variation analysis (GSEA and GSVA) were performed to identify the potential pathways and functions of BRIP1. Besides, BRIP1 correlations with tumor microenvironment (TME), immune infiltration, immune-related genes, tumor mutation burden (TMB), microsatellite instability (MSI), and immunotherapy as well as antitumor drugs were explored in pan-cancer. Differential analyses showed an increased expression of BRIP1 in 28 cancer types and its aberrant expression could be an indicator for prognosis in most cancers. Among the various mutation types of BRIP1 in pan-cancer, amplification was the most common type. BRIP1 expression had a significant correlation with CNV and DNA methylation in 23 tumor types and 16 tumor types, respectively. PPI, GSEA, and GSVA results validated the association between BRIP1 and DNA damage and repair, cell cycle, and metabolism. In addition, the expression of BRIP1 and its correlation with TME, immune-infiltrating cells, immune-related genes, TMB, and MSI as well as a variety of antitumor drugs and immunotherapy were confirmed. Our study indicates that BRIP1 plays an imperative role in the tumorigenesis and immunity of various tumors. It may not only serve as a diagnostic and prognostic biomarker but also can be a predictor for drug sensitivity and immunoreaction during antitumor treatment in pan-cancer.

Transcriptome-wide analysis reveals core transcriptional regulators associated with culm development and variation in Dendrocalamus sinicus, the strongest woody bamboo in the world

Transcription factors (TFs) play indispensable roles in plant development and stress responses. As the largest woody bamboo species in the world, Dendrocalamus sinicus is endemic to Yunnan Province, China, and possesses two natural variants characterized by culm shape, namely straight or bent culms. Understanding the transcriptional regulation network of D. sinicus provides a unique opportunity to clarify the growth and development characteristics of woody bamboos. In this study, 10,236 TF transcripts belonging to 57 families were identified from transcriptome data of two variants at different developmental stages, from which we constructed a transcriptional regulatory network and unigene-coding protein-TFs interactive network of culm development for this attractive species. Gene function enrichment analysis revealed that hormone signaling and MAPK signaling pathways were two most enriched pathways in TF-regulated network. Based on PPI analysis, 50 genes interacting with nine TFs were screened as the core regulation components related to culm development. Of them, 18 synergistic genes of seven TFs, including nuclear cap-binding protein subunit 1, transcription factor GTE9-like, and ATP-dependent DNA helicase DDX11 isoform X1, involved in culm-shape variation. Most of these genes would interact with MYB, C3H, and ARF transcription factors. Six members with two each from ARF, C3H, and MYB transcription factor families and six key interacting genes (IAA3, IAA19, leucine-tRNA ligase, nuclear cap-binding protein subunit 1, elongation factor 2, and coiled-coil domain-containing protein 94) cooperate with these transcription factors were differentially expressed at development stage of young culms, and were validated by quantitative PCR. Our results represent a crucial step towards understanding the regulatory mechanisms of TFs involved in culm development and variation of D. sinicus.

Identification of a novel target of SETD1A histone methyltransferase and the clinical significance in pancreaticcancer.

Although histone H3K4 methyltransferase SETD1A is overexpressed in various cancer types, the molecular mechanism underlying its overexpression and its target genes in pancreatic ductal adenocarcinoma (PDAC) remain unclarified. We conducted immunohistochemical staining for SETD1A in 105 human PDAC specimens to assess the relationship between SETD1A overexpression and clinicopathological features. The function and target genes of SETD1A were investigated using human pancreatic cancer cell lines. SETD1A expression was upregulated in 51.4% of patients with PDAC and was an independent prognostic factor associated with shorter disease-free survival after resection (p< 0.05). Knockdown and overexpression of SETD1A showed that SETD1A plays a crucial role in increasing the proliferation and motility of PDAC cells. SETD1A overexpression increased tumorigenicity. RNA sequencing of SETD1A-knockdown cells revealed downregulation of RUVBL1, an oncogenic protein ATP-dependent DNA helicase gene. ChIP analysis revealed that SETD1A binds to the RUVBL1 promoter region, resulting in increased H3K4me3 levels. Knockdown of RUVBL1 showed inhibition of cell proliferation, migration, and invasion of PDAC cells, which are similar biological effects to SETD1A knockdown. High expression of both SETD1A and RUVBL1 was an independent prognostic factor not only for disease-free survival but also for overall survival (p< 0.05). In conclusion, we identified RUVBL1 as a novel downstream target gene of the SETD1A-H3K4me3 pathway. Co-expression of SETD1A and RUVBL1 is an important factor for predicting the prognosis of patients with PDAC.

Structure-function analysis of DEAD-box helicase DDX43.

DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N terminus, a helicase core domain in its C terminus, and a flexible linker domain in between. DDX43 expression is low or undetectable in normal tissue, but is overexpressed in many tumors; therefore, it is considered a potential target molecule for cancer therapy. We, along with other groups, have shown that DDX43 is an ATP-dependent RNA and DNA helicase, and the KH domain is required for its ATPase and unwinding activity. Electrophoretic mobility shift assay (EMSA), SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, crosslinking immunoprecipitation (CLIP)-seq, and nuclear magnetic resonance (NMR) showed that the KH domain prefers to bind pyrimidine-rich ssDNA and ssRNA, such as TTGT in the promoter regions of genes. Moreover, the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase. No animal model has been generated for DDX43; cellular studies have revealed that DDX43 has roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. Structural and functional studies of DDX43 will not only advance our understanding of DEAD-box helicases and KH domains, but also shed light on the application of DDX43 as therapeutics, where its key binding sites can be targeted by small molecules and natural products as an alternative approach in treating DDX43 overexpressed cancers.

Molecular events confirming antimutagenicity to abscisic acid derived from a floral honey establishing its functional relevance

Natural dietary products of health promoting and disease preventive functional relevance are gaining significant prominence. Current investigation was aimed to decipher the underlying molecular mechanism responsible for the antimutagenic action contributing to functional relevance of floral honey (‘Pongammia pinnata’, Karanj honey) derived abscisic acid (ABA) against ethyl methanesulfonate (EMS) induced mutagenesis. Differential expression of proteins under different treatment conditions was ascertained by 2D gel electrophoresis. Selectively up-regulated characterized using MALDI-TOF MS/MS were identified as polyribonucleotide nucleotidyl transferse (PNPase), LPS-assembly lipoprotein (LptE), Outer membrane Usher protein (HtrE), ATP-dependent DNA helicase (RecG), and Phosphomethyl pyrimidine synthase (ThiC). Antimutagenicity exerted by ABA against EMS was ∼78% in wild type E. coli MG1655 strain however, in E. coli MG1655 ΔthiC, ΔpnpA, ΔrecG, and ΔhtrE this activity was found to be ∼60, 10, 9 and 10%, respectively. Proteomic analysis and antimutagenicity studies using E. coli single gene knockout strains thus indicated about the possible role of thiC, htrE, lptE, recG and pnp in observed antimutagenicity. Cyclic voltametry as well as competition kinetics through pulse radiolysis confirmed lack of antioxidant capacity in abscisic acid apparently ruling out the possibility of scavenging of electrophilic intermediates generated by ethyl methanesulfonate. It is proposed that ABA is exerting antimutagenicity through its involvement at the cellular level leading to physiological adaptation, strengthening of cell wall proteins and up-regulation of the repair proteins. This study provides a novel dimension to the functional role of abscisic acid from its nutraceutical perspective.

Chl1, an ATP-Dependent DNA Helicase, Inhibits DNA:RNA Hybrids Formation at DSB Sites to Maintain Genome Stability in S. pombe.

As an ATP-dependent DNA helicase, human ChlR1/DDX11 (Chl1 in yeast) can unwind both DNA:RNA and DNA:DNA substrates in vitro. Studies have demonstrated that ChlR1 plays a vital role in preserving genome stability by participating in DNA repair and sister chromatid cohesion, whereas the ways in which the biochemical features of ChlR1 function in DNA metabolism are not well understood. Here, we illustrate that Chl1 localizes to double-strand DNA break (DSB) sites and restrains DNA:RNA hybrid accumulation at these loci. Mutation of Chl1 strongly impairs DSB repair capacity by homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways, and deleting RNase H further reduces DNA repair efficiency, which indicates that the enzymatic activities of Chl1 are needed in Schizosaccharomyces pombe. In addition, we found that the Rpc37 subunit of RNA polymerase III (RNA Pol III) interacts directly with Chl1 and that deletion of Chl1 has no influence on the localization of Rpc37 at DSB site, implying the role of Rpc37 in the recruitment of Chl1 to this site.

ATP-dependent DNA helicase (TaDHL), a Novel Reduced-Height (Rht) Gene in Wheat.

In wheat, a series of dwarf and semi-dwarf plant varieties have been developed and utilized worldwide since the 1960s and caused the ‘Green Revolution’. To date, 25 reduced-height (Rht) genes have been identified, but only several genes for plant height (PH) have been isolated previously. In this study, we identified a candidate gene, ATP-dependent DNA helicase (TaDHL-7B), for PH via QTL mapping and genome-wide association study (GWAS) methods. We knocked out this gene using the CRISPR/Cas9 system in variety ‘Fielder’. Two homozygous mutant genotypes, AAbbDD (−5 bp) and AAbbDD (−1 bp), were obtained in the T2 generation. The PH values of AAbbDD (−5 bp) and AAbbDD (−1 bp) were significantly reduced compared with the wild-type (WT, ‘Fielder’), indicating that TaDHL-7B is a novel Rht gene that controls the PH. This is the first time that a PH gene of wheat has been isolated with a non-hormone pathway, providing a new insight into the genetic control of PH. The TaDHL gene reduced the PH without a yield penalty. It could be used to improve the lodging resistance and yield in wheat breeding programs.

Searching for the Roots of Bloom Syndrome Protein and Its Homologs Using Phylogenetic Analysis

Phylogenetic analysis (PA) is used for elucidation of relationships among different species and provides information about their evolution. BLM protein (BLM RecQ like helicase) is responsible for the repair of stalled replication fork during double-strand break repair by homologous recombination. In the current study, phylogenetic analysis was performed using BLM protein sequences, sequences of its homologs and its putative homologs from 34 species including covering the genera of Bacteria, Archaea and Eukaryotes. This study was carried out for the purpose of (1) illustrating and comparing relationships among eukaryotic BLM proteins, their homologs (ATP-dependent DNA helicase RecQs in Bacteria) and their potential putative homologs (ATP-dependent DNA helicase Hel308s in Archaea), (2) evaluating how BLM protein evolution took place, what it brought to the organisms by acquiring functional changes and how future potential changes would occur and (3) gaining the general perspective in the evolution of BLM protein. All analyzed species in Bacteria, Archaea and Eukaryota formed a clear inter-species cluster, except for P. sinensis (Reptilia). The results supported that Hjm helicase may be one of the candidate potential ancestors of the BLM proteins and their homologs. Moreover, especially two domains which are Helicase ATP-binding and Helicase C-terminal domain were encountered in the all analyzed species and seem to be strictly conserved in the future. Repair related-highly sophisticated interaction network of BLM indicated that its functional evolution reaches a certain level and it appears to have taken an important place in maintaining genomic stability. However, it should be taken into account that BLM may acquire additional functions or become a cornerstone in different pathways in the future depending on its participation in various metabolic roads.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Molecular Mechanisms of the RECQ4 Pathogenic Mutations.

The human RECQ4 gene encodes an ATP-dependent DNA helicase that contains a conserved superfamily II helicase domain located at the center of the polypeptide. RECQ4 is one of the five RECQ homologs in human cells, and its helicase domain is flanked by the unique amino and carboxyl termini with sequences distinct from other members of the RECQ helicases. Since the identification of the RECQ4 gene in 1998, multiple RECQ4 mutations have been linked to the pathogenesis of three clinical diseases, which are Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO. Patients with these diseases show various developmental abnormalities. In addition, a subset of RECQ4 mutations are associated with high cancer risks, especially for osteosarcoma and/or lymphoma at early ages. The discovery of clinically relevant RECQ4 mutations leads to intriguing questions: how is the RECQ4 helicase responsible for preventing multiple clinical syndromes? What are the mechanisms by which the RECQ4 disease mutations cause tissue abnormalities and drive cancer formation? Furthermore, RECQ4 is highly overexpressed in many cancer types, raising the question whether RECQ4 acts not only as a tumor suppressor but also an oncogene that can be a potential new therapeutic target. Defining the molecular dysfunctions of different RECQ4 disease mutations is imperative to improving our understanding of the complexity of RECQ4 clinical phenotypes and the dynamic roles of RECQ4 in cancer development and prevention. We will review recent progress in examining the molecular and biochemical properties of the different domains of the RECQ4 protein. We will shed light on how the dynamic roles of RECQ4 in human cells may contribute to the complexity of RECQ4 clinical phenotypes.

Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage

Shigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.

Gain-of-function mutagenesis through activation tagging identifies XPB2 and SEN1 helicase genes as potential targets for drought stress tolerance in rice.

XPB2 and SEN1 helicases were identified through activation tagging as potential candidate genes in rice for inducing high water-use efficiency (WUE) and maintaining sustainable yield under drought stress. As a follow-up on the high-water-use-efficiency screening and physiological analyses of the activation-tagged gain-of-function mutant lines that were developed in an indica rice variety, BPT-5204 (Moin et al. in Plant Cell Environ 39:2440-2459, 2016a, https://doi.org/10.1111/pce.12796 ), we have identified two gain-of-function mutant lines (XM3 and SM4), which evidenced the activation of two helicases, ATP-dependent DNA helicase (XPB2) and RNA helicase (SEN1), respectively. We performed the transcript profiling of XPB2 and SEN1 upon exposure to various stress conditions and found their significant upregulation, particularly in ABA and PEG treatments. Extensive morpho-physiological and biochemical analyses based on 24 metrics were performed under dehydration stress (PEG) and phytohormone (ABA) treatments for the wild-type and the two mutant lines. Principal component analysis (PCA) performed on the dataset captured 72.73% of the cumulative variance using the parameters influencing the first two principal components. The tagged mutants exhibited reduced leaf wilting, improved revival efficiency, constant amylose:amylopectin ratio, high chlorophyll and proline contents, profuse tillering, high quantum efficiency and yield-related traits with respect to their controls. These observations were further validated under greenhouse conditions by the periodic withdrawal of water atthe pot level. Germination of the seeds of these mutant lines indicated their insensitivity to high ABA concentration. The associated upregulation of stress-specific genes further suggests that their drought tolerance might be because of the coordinated expression of several stress-responsive genes in these two mutants. Altogether, our results provided a firm basis for SEN1 and XPB2 as potential candidates for manipulation of drought tolerance and improving rice performance and yield under limited water conditions.

Functions of BLM Helicase in Cells: Is It Acting Like a Double-Edged Sword?

DNA damage repair response is an important biological process involved in maintaining the fidelity of the genome in eukaryotes and prokaryotes. Several proteins that play a key role in this process have been identified. Alterations in these key proteins have been linked to different diseases including cancer. BLM is a 3′−5′ ATP-dependent RecQ DNA helicase that is one of the most essential genome stabilizers involved in the regulation of DNA replication, recombination, and both homologous and non-homologous pathways of double-strand break repair. BLM structure and functions are known to be conserved across many species like yeast, Drosophila, mouse, and human. Genetic mutations in the BLM gene cause a rare, autosomal recessive disorder, Bloom syndrome (BS). BS is a monogenic disease characterized by genomic instability, premature aging, predisposition to cancer, immunodeficiency, and pulmonary diseases. Hence, these characteristics point toward BLM being a tumor suppressor. However, in addition to mutations, BLM gene undergoes various types of alterations including increase in the copy number, transcript, and protein levels in multiple types of cancers. These results, along with the fact that the lack of wild-type BLM in these cancers has been associated with increased sensitivity to chemotherapeutic drugs, indicate that BLM also has a pro-oncogenic function. While a plethora of studies have reported the effect of BLM gene mutations in various model organisms, there is a dearth in the studies undertaken to investigate the effect of its oncogenic alterations. We propose to rationalize and integrate the dual functions of BLM both as a tumor suppressor and maybe as a proto-oncogene, and enlist the plausible mechanisms of its deregulation in cancers.

Abstract 1768: Identification of a potent, orally-available SMARCA2/4 inhibitor with in vitro and in vivo activity in preclinical models of SMARCA4-mutant NSCLC

Abstract SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) is an ATP-dependent DNA helicase and a catalytic component of the SWI/SNF complex. SWI/SNF-mediated nucleosome remodeling is a critical regulator of chromatin accessibility resulting in transcriptional regulation of gene sets that determine and maintain cell state. The role of SMARCA2 as the catalytic driver of SWI/SNF activity is mutually exclusive with that of its close paralog, SMARCA4. Loss of functional SMARCA4, as is reported in a subset of NSCLC and other cancer types generally confers a dependency on SMARCA2. Cancers lacking functional SMARCA4 have been shown to be sensitive to loss of SMARCA2 function either through genetic ablation or the use of a small molecule SMARCA2/4 inhibitor. We describe the identification and characterization of a small molecule SMARCA2/4 inhibitor Compound 1 and its selective activity in the SMARCA4-mutant setting both in in vitro and in vivo model systems. The compound demonstrates inhibition of SMARCA2 and SMARCA4 enzymatic activity in biochemical assays and broad selectivity against other helicases. Selective sensitivity in anti-proliferative assays of 15-fold was observed in NSCLC cell lines with SMARCA4 protein loss compared to those harboring WT SMARCA4 protein. Oral dosing demonstrates dose-dependent in vivo SMARCA2 inhibition and anti-tumor activity in SMARCA4-mutant NSCLC xenograft models. This compound is suitable for further exploration of the role of SMARCA2/4 and SWI/SNF in vitro and in vivo in cancer and other indications. Citation Format: Allison E. Drew, Suzanne L. Jacques, Lindsey W. Eichinger, Chloe Pantano, Vinny Motwani, Cuyue Tang, Cuyue Tang, Neil Farrow, Dorothy Brach, Selene Howe, Alejandra Raimondi, Daniel T. Dransfield, Kenneth W. Duncan, Kim Stickland, Liyue Huang, John Lampe. Identification of a potent, orally-available SMARCA2/4 inhibitor with in vitro and in vivo activity in preclinical models of SMARCA4-mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1768.