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1
Sun, Zhi-Yin, Xiao-Na Wang, Sui-Qi Cheng, Xiao-Xuan Su, and Tian-Miao Ou. "Developing Novel G-Quadruplex Ligands: from Interaction with Nucleic Acids to Interfering with Nucleic Acid–Protein Interaction." Molecules 24, no. 3 (January 22, 2019): 396. http://dx.doi.org/10.3390/molecules24030396.
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G-quadruplex is a special secondary structure of nucleic acids in guanine-rich sequences of genome. G-quadruplexes have been proved to be involved in the regulation of replication, DNA damage repair, and transcription and translation of oncogenes or other cancer-related genes. Therefore, targeting G-quadruplexes has become a novel promising anti-tumor strategy. Different kinds of small molecules targeting the G-quadruplexes have been designed, synthesized, and identified as potential anti-tumor agents, including molecules directly bind to the G-quadruplex and molecules interfering with the binding between the G-quadruplex structures and related binding proteins. This review will explore the feasibility of G-quadruplex ligands acting as anti-tumor drugs, from basis to application. Meanwhile, since helicase is the most well-defined G-quadruplex-related protein, the most extensive research on the relationship between helicase and G-quadruplexes, and its meaning in drug design, is emphasized.
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Wu, Yuliang, Kazuo Shin-ya, and Robert M. Brosh. "FANCJ Helicase Defective in Fanconia Anemia and Breast Cancer Unwinds G-Quadruplex DNA To Defend Genomic Stability." Molecular and Cellular Biology 28, no. 12 (April 21, 2008): 4116–28. http://dx.doi.org/10.1128/mcb.02210-07.
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ABSTRACT FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.
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Shukla, Kaustubh, Roshan Singh Thakur, Debayan Ganguli, Desirazu Narasimha Rao, and Ganesh Nagaraju. "Escherichia coli and Neisseria gonorrhoeae UvrD helicase unwinds G4 DNA structures." Biochemical Journal 474, no. 21 (October 18, 2017): 3579–97. http://dx.doi.org/10.1042/bcj20170587.
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G-quadruplex (G4) secondary structures have been implicated in various biological processes, including gene expression, DNA replication and telomere maintenance. However, unresolved G4 structures impede replication progression which can lead to the generation of DNA double-strand breaks and genome instability. Helicases have been shown to resolve G4 structures to facilitate faithful duplication of the genome. Escherichia coli UvrD (EcUvrD) helicase plays a crucial role in nucleotide excision repair, mismatch repair and in the regulation of homologous recombination. Here, we demonstrate a novel role of E. coli and Neisseria gonorrhoeae UvrD in resolving G4 tetraplexes. EcUvrD and N. gonorrhoeae UvrD were proficient in unwinding previously characterized tetramolecular G4 structures. Notably, EcUvrD was equally efficient in resolving tetramolecular and bimolecular G4 DNA that were derived from the potential G4-forming sequences from the genome of E. coli. Interestingly, in addition to resolving intermolecular G4 structures, EcUvrD was robust in unwinding intramolecular G4 structures. These data for the first time provide evidence for the role of UvrD in the resolution of G4 structures, which has implications for the in vivo role of UvrD helicase in G4 DNA resolution and genome maintenance.
4
Bryan, Tracy M. "Mechanisms of DNA Replication and Repair: Insights from the Study of G-Quadruplexes." Molecules 24, no. 19 (September 22, 2019): 3439. http://dx.doi.org/10.3390/molecules24193439.
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G-quadruplexes are four-stranded guanine-rich structures that have been demonstrated to occur across the genome in humans and other organisms. They provide regulatory functions during transcription, translation and immunoglobulin gene rearrangement, but there is also a large amount of evidence that they can present a potent barrier to the DNA replication machinery. This mini-review will summarize recent advances in understanding the many strategies nature has evolved to overcome G-quadruplex-mediated replication blockage, including removal of the structure by helicases or nucleases, or circumventing the deleterious effects on the genome through homologous recombination, alternative end-joining or synthesis re-priming. Paradoxically, G-quadruplexes have also recently been demonstrated to provide a positive role in stimulating the initiation of DNA replication. These recent studies have not only illuminated the many roles and consequences of G-quadruplexes, but have also provided fundamental insights into the general mechanisms of DNA replication and its links with genetic and epigenetic stability.
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Byrd, Alicia K., and Kevin D. Raney. "Structure and function of Pif1 helicase." Biochemical Society Transactions 45, no. 5 (September 12, 2017): 1159–71. http://dx.doi.org/10.1042/bst20170096.
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Pif1 family helicases have multiple roles in the maintenance of nuclear and mitochondrial DNA in eukaryotes. Saccharomyces cerevisiae Pif1 is involved in replication through barriers to replication, such as G-quadruplexes and protein blocks, and reduces genetic instability at these sites. Another Pif1 family helicase in S. cerevisiae, Rrm3, assists in fork progression through replication fork barriers at the rDNA locus and tRNA genes. ScPif1 (Saccharomyces cerevisiae Pif1) also negatively regulates telomerase, facilitates Okazaki fragment processing, and acts with polymerase δ in break-induced repair. Recent crystal structures of bacterial Pif1 helicases and the helicase domain of human PIF1 combined with several biochemical and biological studies on the activities of Pif1 helicases have increased our understanding of the function of these proteins. This review article focuses on these structures and the mechanism(s) proposed for Pif1's various activities on DNA.
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Schwab, Rebekka A., Jadwiga Nieminuszczy, Kazuo Shin-ya, and Wojciech Niedzwiedz. "FANCJ couples replication past natural fork barriers with maintenance of chromatin structure." Journal of Cell Biology 201, no. 1 (March 25, 2013): 33–48. http://dx.doi.org/10.1083/jcb.201208009.
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Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer–predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.
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Lowran, Kaitlin, Laura Campbell, Phillip Popp, and Colin G. Wu. "Assembly of a G-Quadruplex Repair Complex by the FANCJ DNA Helicase and the REV1 Polymerase." Genes 11, no. 1 (December 19, 2019): 5. http://dx.doi.org/10.3390/genes11010005.
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The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.
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Wu, Yuliang, Joshua A. Sommers, Avvaru N. Suhasini, Thomas Leonard, Julianna S. Deakyne, Alexander V. Mazin, Kazuo Shin-ya, Hiroyuki Kitao, and Robert M. Brosh. "Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes." Blood 116, no. 19 (November 11, 2010): 3780–91. http://dx.doi.org/10.1182/blood-2009-11-256016.
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Abstract Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.
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Gaur, Paras, Fletcher E. Bain, Masayoshi Honda, Sophie L. Granger, and Maria Spies. "Single-Molecule Analysis of the Improved Variants of the G-Quadruplex Recognition Protein G4P." International Journal of Molecular Sciences 24, no. 12 (June 17, 2023): 10274. http://dx.doi.org/10.3390/ijms241210274.
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As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine–guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein’s affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.
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Ononye, Onyekachi E., Christopher W. Sausen, Lata Balakrishnan, and Matthew L. Bochman. "Lysine acetylation regulates the activity of nuclear Pif1." Journal of Biological Chemistry 295, no. 46 (September 2, 2020): 15482–97. http://dx.doi.org/10.1074/jbc.ra120.015164.
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In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo. Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.
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Cueny, Rachel R., Sameer Varma, Kristina H. Schmidt, and James L. Keck. "Biochemical properties of naturally occurring human bloom helicase variants." PLOS ONE 18, no. 6 (June 2, 2023): e0281524. http://dx.doi.org/10.1371/journal.pone.0281524.
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Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636–1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.
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White, Malcolm F. "Structure, function and evolution of the XPD family of iron–sulfur-containing 5′→3′ DNA helicases." Biochemical Society Transactions 37, no. 3 (May 20, 2009): 547–51. http://dx.doi.org/10.1042/bst0370547.
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The XPD (xeroderma pigmentosum complementation group D) helicase family comprises a number of superfamily 2 DNA helicases with members found in all three domains of life. The founding member, the XPD helicase, is conserved in archaea and eukaryotes, whereas the closest homologue in bacteria is the DinG (damage-inducible G) helicase. Three XPD paralogues, FancJ (Fanconi's anaemia complementation group J), RTEL (regular of telomere length) and Chl1, have evolved in eukaryotes and function in a variety of DNA recombination and repair pathways. All family members are believed to be 5′→3′ DNA helicases with a structure that includes an essential iron–sulfur-cluster-binding domain. Recent structural, mutational and biophysical studies have provided a molecular framework for the mechanism of the XPD helicase and help to explain the phenotypes of a considerable number of mutations in the XPD gene that can cause three different genetic conditions: xeroderma pigmentosum, trichothiodystrophy and Cockayne's syndrome. Crystal structures of XPD from three archaeal organisms reveal a four-domain structure with two canonical motor domains and two unique domains, termed the Arch and iron–sulfur-cluster-binding domains. The latter two domains probably collaborate to separate duplex DNA during helicase action. The role of the iron–sulfur cluster and the evolution of the XPD helicase family are discussed.
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Huang, Joe Chin-Sun, Julia Sidorova, Sylvia Chien, Jin Dai, Ben Logsdon, Su-In Lee, Raymond J. Monnat, and Pamela S. Becker. "Mini-Chromosome Maintenance (MCM) DNA Helicase Genes Influence Acute Myeloid Leukemia (AML) Replication and Response to Chemotherapy-Induced DNA Damage." Blood 126, no. 23 (December 3, 2015): 3629. http://dx.doi.org/10.1182/blood.v126.23.3629.3629.
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Abstract Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic stem/progenitor cells (HSCs). For older adults (≥60 yo) with AML, the prognosis is poor. The functional decline of HSCs with aging has been linked to increased replication stress from decreased expression of mini-chromosome maintenance (MCM) DNA helicase complex proteins. As AML incidence sharply rises with age, we explored age-related differences in gene expression of MCM and RECQ DNA helicases and DNA damage response (DDR) genes in AML patient blood and bone marrow samples. We hypothesized that older AML patients would show differences in DNA replication and DDR pathways compared to younger patients. We began with an analysis of the TCGA AML database for MCM and RECQ helicase gene aberrations and found these in 37% (61/166) of the cases, with a median age of 60 years. There was reduced 5-year overall survival, 8.2 vs. 26.3 months, for those with vs. without such defects. Only a few mutations occur in these genes, with majority of aberrations due to mRNA up-regulation. 30 AML patient samples were obtained at diagnosis (n = 24) or relapse (n = 6). Total RNA was extracted from AML blasts and gene expression examined with the Affymetrix U133 Plus 2 arrays. Patients were categorized as older (≥65 yo), middle-aged (50-64 yo) and younger (<50 yo). Ingenuity Pathway Analysis (IPA) was performed on differentially expressed genes between the age-groups. FACS was used to analyze the effect of cytokine stimulation (G-CSF, IL-3, SCF) +/- 5 µM mitomycin C (induces double stranded DNA breaks) on levels of BrdU, γ-H2AX and cleaved-PARP as measures of cell cycle activity, DNA damage and apoptosis, respectively, in AML blasts. Older AML patients showed increased expression of the MCM and RECQ DNA helicases and in multiple genes involved in the DDR response, including Ataxia telangiectasia mutated (ATM)/ATM- and RAD3-related (ATR) signaling, homologous recombination (HR), nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and Fanconi anemia (FA) families. Increased expression of the MCM, RECQ helicase and DDR genes in AML blast cells conferred a significantly worse prognosis. Basal γ-H2AX levels were increased in AML patients with abnormal or complex karyotype vs. normal karyotype. IPA showed age-related changes in ERG transcription factor activity, NFκB signaling and histone H3 modification. AML cells with high vs. low MCM gene expression differed in their response to growth factor stimulation and MMC treatment, in that the low MCM3 gene expressors did not progress through cell cycle after treatment with myeloid growth factors, and thus were spared of DNA damage and induction of apoptosis with MMC treatment. By Western blot analysis compared to actin control, the low MCM3 gene expressors also exhibited a trend toward significant positive correlation with MCM3 protein levels (Pearson r = 0.7, p = 0.06) In summary, older AML patients exhibited increased expression of MCM and RECQ helicases and multiple DDR genes compared to middle-aged and younger patients, and there were age-associated changes in ERG transcriptional activity, NFkB signaling and histone H3 epigenetic regulation of gene expression. These elements are thus potential targets for future drug development, particularly for older adults with AML. Disclosures No relevant conflicts of interest to declare.
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Youds, Jillian L., Louise J. Barber, Jordan D. Ward, Spencer J. Collis, Nigel J. O'Neil, Simon J. Boulton, and Ann M. Rose. "DOG-1 Is the Caenorhabditis elegans BRIP1/FANCJ Homologue and Functions in Interstrand Cross-Link Repair." Molecular and Cellular Biology 28, no. 5 (December 17, 2007): 1470–79. http://dx.doi.org/10.1128/mcb.01641-07.
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ABSTRACT Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.
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Komůrková, Denisa, Alena Svobodová Kovaříková, and Eva Bártová. "G-Quadruplex Structures Colocalize with Transcription Factories and Nuclear Speckles Surrounded by Acetylated and Dimethylated Histones H3." International Journal of Molecular Sciences 22, no. 4 (February 17, 2021): 1995. http://dx.doi.org/10.3390/ijms22041995.
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G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.
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Aklilu, Behailu B., François Peurois, Carole Saintomé, Kevin M. Culligan, Daniela Kobbe, Catherine Leasure, Michael Chung, et al. "Functional Diversification of Replication Protein A Paralogs and Telomere Length Maintenance in Arabidopsis." Genetics 215, no. 4 (June 12, 2020): 989–1002. http://dx.doi.org/10.1534/genetics.120.303222.
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Replication protein A (RPA) is essential for many facets of DNA metabolism. The RPA gene family expanded in Arabidopsis thaliana with five phylogenetically distinct RPA1 subunits (RPA1A-E), two RPA2 (RPA2A and B), and two RPA3 (RPA3A and B). RPA1 paralogs exhibit partial redundancy and functional specialization in DNA replication (RPA1B and RPA1D), repair (RPA1C and RPA1E), and meiotic recombination (RPA1A and RPA1C). Here, we show that RPA subunits also differentially impact telomere length set point. Loss of RPA1 resets bulk telomeres at a shorter length, with a functional hierarchy for replication group over repair and meiosis group RPA1 subunits. Plants lacking RPA2A, but not RPA2B, harbor short telomeres similar to the replication group. Telomere shortening does not correlate with decreased telomerase activity or deprotection of chromosome ends in rpa mutants. However, in vitro assays show that RPA1B2A3B unfolds telomeric G-quadruplexes known to inhibit replications fork progression. We also found that ATR deficiency can partially rescue short telomeres in rpa2a mutants, although plants exhibit defects in growth and development. Unexpectedly, the telomere shortening phenotype of rpa2a mutants is completely abolished in plants lacking the RTEL1 helicase. RTEL1 has been implicated in a variety of nucleic acid transactions, including suppression of homologous recombination. Thus, the lack of telomere shortening in rpa2a mutants upon RTEL1 deletion suggests that telomere replication defects incurred by loss of RPA may be bypassed by homologous recombination. Taken together, these findings provide new insight into how RPA cooperates with replication and recombination machinery to sustain telomeric DNA.
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Sanders, Cyril M. "Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity." Biochemical Journal 430, no. 1 (July 28, 2010): 119–28. http://dx.doi.org/10.1042/bj20100612.
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Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G4 (G-quadruplex) structures by acting as a G4 resolvase. The present study shows that human Pif1 (hPif1, nuclear form) is a G4 DNA-binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206–620 of 641, hPifHD). hPif1 preferentially bound synthetic G4 DNA relative to ssDNA (single-stranded DNA), dsDNA (double-stranded DNA) and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding, but not binding, required an extended (>10 nucleotide) 5′ ssDNA tail, and in competition assays, G4 DNA was an ineffective suppressor of helicase activity compared with ssDNA. These results suggest a distinction between the determinants of G4 DNA binding and the ssDNA interactions required for helicase action and that hPif1 may act on G4 substrates by binding alone or as a resolvase. Human Pif1 could therefore have a role in processing G4 structures that arise in the single-stranded nucleic acid intermediates formed during DNA replication and gene expression.
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Byrd, Alicia K., Matthew R. Bell, and Kevin D. Raney. "Pif1 helicase unfolding of G-quadruplex DNA is highly dependent on sequence and reaction conditions." Journal of Biological Chemistry 293, no. 46 (September 26, 2018): 17792–802. http://dx.doi.org/10.1074/jbc.ra118.004499.
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In addition to unwinding double-stranded nucleic acids, helicase activity can also unfold noncanonical structures such as G-quadruplexes. We previously characterized Pif1 helicase catalyzed unfolding of parallel G-quadruplex DNA. Here we characterized unfolding of the telomeric G-quadruplex, which can fold into antiparallel and mixed hybrid structures and found significant differences. Telomeric DNA sequences are unfolded more readily than the parallel quadruplex formed by the c-MYC promoter in K+. Furthermore, we found that under conditions in which the telomeric quadruplex is less stable, such as in Na+, Pif1 traps thermally melted quadruplexes in the absence of ATP, leading to the appearance of increased product formation under conditions in which the enzyme is preincubated with the substrate. Stable telomeric G-quadruplex structures were unfolded in a stepwise manner at a rate slower than that of duplex DNA unwinding; however, the slower dissociation from G-quadruplexes compared with duplexes allowed the helicase to traverse more nucleotides than on duplexes. Consistent with this, the rate of ATP hydrolysis on the telomeric quadruplex DNA was reduced relative to that on single-stranded DNA (ssDNA), but less quadruplex DNA was needed to saturate ATPase activity. Under single-cycle conditions, telomeric quadruplex was unfolded by Pif1, but for the c-MYC quadruplex, unfolding required multiple helicase molecules loaded onto the adjacent ssDNA. Our findings illustrate that Pif1-catalyzed unfolding of G-quadruplex DNA is highly dependent on the specific sequence and the conditions of the reaction, including both the monovalent cation and the order of addition.
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Shah Punatar, Rajvee, Maria Jose Martin, Haley D. M. Wyatt, Ying Wai Chan, and Stephen C. West. "Resolution of single and double Holliday junction recombination intermediates by GEN1." Proceedings of the National Academy of Sciences 114, no. 3 (January 3, 2017): 443–50. http://dx.doi.org/10.1073/pnas.1619790114.
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Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5′-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase,Escherichia coliRuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5′-A/TTTG/C-3′.
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Krzeptowski, Wojciech, Patryk Chudy, Grzegorz Sokołowski, Monika Żukowska, Anna Kusienicka, Agnieszka Seretny, Agata Kalita, et al. "Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?" Antioxidants 10, no. 1 (January 12, 2021): 94. http://dx.doi.org/10.3390/antiox10010094.
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G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.
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Lerner, Leticia Koch, and Julian E. Sale. "Replication of G Quadruplex DNA." Genes 10, no. 2 (January 29, 2019): 95. http://dx.doi.org/10.3390/genes10020095.
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A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.
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Kim, Jung Min, Younghoon Kee, Allan Gurtan, and Alan D. D'Andrea. "Cell cycle–dependent chromatin loading of the Fanconi anemia core complex by FANCM/FAAP24." Blood 111, no. 10 (May 15, 2008): 5215–22. http://dx.doi.org/10.1182/blood-2007-09-113092.
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Abstract Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. A total of 13 FA proteins are involved in regulating genome surveillance and chromosomal stability. The FA core complex, consisting of 8 FA proteins (A/B/C/E/F/G/L/M), is essential for the monoubiquitination of FANCD2 and FANCI. FANCM is a human ortholog of the archaeal DNA repair protein Hef, and it contains a DEAH helicase and a nuclease domain. Here, we examined the effect of FANCM expression on the integrity and localization of the FA core complex. FANCM was exclusively localized to chromatin fractions and underwent cell cycle–dependent phosphorylation and dephosphorylation. FANCM-depleted HeLa cells had an intact FA core complex but were defective in chromatin localization of the complex. Moreover, depletion of the FANCM binding partner, FAAP24, disrupted the chromatin association of FANCM and destabilized FANCM, leading to defective recruitment of the FA core complex to chromatin. Our results suggest that FANCM is an anchor required for recruitment of the FA core complex to chromatin, and that the FANCM/FAAP24 interaction is essential for this chromatin-loading activity. Dysregulated loading of the FA core complex accounts, at least in part, for the characteristic cellular and developmental abnormalities in FA.
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Tuesuwan, Bodin, Jonathan T. Kern, Pei Wang Thomas, Mireya Rodriguez, Jing Li, Wendi M. David, and Sean M. Kerwin. "Simian Virus 40 Large T-Antigen G-Quadruplex DNA Helicase Inhibition by G-Quadruplex DNA-Interactive Agents†." Biochemistry 47, no. 7 (February 2008): 1896–909. http://dx.doi.org/10.1021/bi701747d.
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Truong, Tuom TT, Trang PT Phan, Linh TT Le, Dung H. Nguyen, Hoang D. Nguyen, and Dung Thanh Dang. "Engineering yellow fluorescent protein probe for visualization of parallel DNA G-quadruplex." Science and Technology Development Journal 21, no. 3 (November 9, 2018): 84–89. http://dx.doi.org/10.32508/stdj.v21i3.461.
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Introduction: The formation of G-quadruplex plays a key role in many biological processes. Therefore, visualization of G-quadruplex is highly essential for design of G-quadruplex-targeted small molecules (drugs). Herein, we report on an engineered fluorescent protein probe which was able to distinguish G-quadruplex topologies.
Methods: The fluorescent protein probe was generated by genetically incorporating yellow fluorescent protein (YFP) to RNA helicase associated with AU-rich element (RHAU) peptide motif.
Results: This probe could selectively bind and visualize parallel G-quadruplex structure (T95-2T) at high affinity (Kd~130 nM). Visualization of the parallel G-quadruplex by RHAU-YFP could be easily observed in vitro by using normal Gel Doc or the naked eye.
Conclusion: The YFP probe could be encoded in cells to provide a powerful tool for detection of parallel G-quadruplexes both in vitro and in vivo.
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Castro, Jennifer, Matthew H. Daniels, Chuang Lu, David Brennan, Deepali Gotur, Young-Tae Lee, Kevin Knockenhauer, et al. "Abstract 1136: Targeting DHX9 inhibition as a novel therapeutic modality in microsatellite instable colorectal cancer." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1136. http://dx.doi.org/10.1158/1538-7445.am2023-1136.
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Abstract DHX9 is a multifunctional DEAH-box ATP-independent RNA helicase which has been reported to play important roles in replication, transcription, translation, RNA splicing and RNA processing which contribute to DHX9’s role in maintenance of genomic stability. Functionally, DHX9’s role involves binding to as well as unwinding and/or resolving double-stranded and single-stranded DNA/RNA, DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G quadraplexes. Overexpression of DHX9 is evident in multiple cancer types, including colorectal cancer (CRC) and lung cancer. In addition, microsatellite instable (MSI) tumors exhibiting defective mismatch repair (dMMR) show a strong dependence on DHX9, making this helicase an attractive target for oncology drug discovery. Here we describe data supporting targeting DHX9 in MSI CRC as a novel therapeutic, and the first identification of potent and selective in vitro and in vivo small molecule inhibitors of DHX9. We demonstrate that DHX9 inhibition in MSI CRC, delivered either through siRNA knockdown or compound treatment, leads to an increase in RNA/DNA secondary structures such as R-loops and circRNA (i.e. circBRIP1) inducing replication stress. Cell lines that are dMMR (i.e. MSI) are unable to resolve this replication stress, resulting in prevention of DNA replication in S phase and later onset of apoptosis. We were able to confirm this selective dependency in a panel of 20 CRC cell lines; anti-proliferative effects mediated by DHX9 inhibition were dependent on cell line dMMR status in a 10-day proliferation assay. Furthermore, compound 1, an orally bioavailable DHX9 inhibitor was used to investigate in vivo efficacy in MSI CRC (LS411N) and MSS CRC (SW480) xenograft models. Compound 1 was well tolerated across the 28-day treatment period with robust and durable tumor regression (TGI = 105 %) observed in the LS411N tumor xenograft model only. In addition, following cessation of treatment, minimal tumor regrowth was observed in a 28-day post treatment window. Tumor and plasma concentrations of compound 1 and changes in pharmacodynamic markers of DHX9 inhibition, such as circBRIP1 mRNA, were measured and resulting PK and PD data were highly correlated. Together, these preclinical data validate DHX9 as a tractable new target with potential utility as a novel treatment for patients with MSI CRC. Citation Format: Jennifer Castro, Matthew H. Daniels, Chuang Lu, David Brennan, Deepali Gotur, Young-Tae Lee, Kevin Knockenhauer, April Case, Jie Wu, Shane M. Buker, Julie Liu, Brian A. Sparling, E. Allen Sickmier, Stephen J. Blakemore, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Scott Ribich, Robert A. Copeland. Targeting DHX9 inhibition as a novel therapeutic modality in microsatellite instable colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1136.
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Wu, Guanhui, Zheng Xing, Luying Chen, Elizabeth J. Tran, and Danzhou Yang. "Abstract 3948: DDX5 helicase resolves G-quadruplex and transactivates MYC expression." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3948. http://dx.doi.org/10.1158/1538-7445.am2023-3948.
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Abstract DDX5 (DEAD-box protein 5) plays an important role in cell proliferation, differentiation, and tumorigenesis. DDX5 is a founding member of the DEAD-box RNA helicase family and known as a non-processive RNA helicase. It acts as a transcriptional co-activator of many cancer-associated genes, such as MYC, however, the underlying molecular mechanism is unknown. MYC is one of the most critical oncogenes and has a DNA G-quadruplex in its proximal promoter region (MycG4) that functions as transcriptional silencer. Guanine-rich DNA and RNA sequences can form G-quadruplex, a non-canonical secondary structure. However, MycG4 is highly stable and its regulatory role in transcription requires active unfolding. Here, we report that DDX5 unfolds MycG4 with extreme efficiency, and thereby transactivates MYC expression. To understand the effects of DDX5 on MycG4, we characterized the unfolding of MycG4-DNA within the DDX5-MycG4 complex using DNA footprinting, FRET and CD spectroscopy. While DDX5 is known as a dsRNA helicase, our results showed that DDX5 is a highly active DNA G4-resolvase. Strikingly, MycG4 unfolding requires neither ATP hydrolysis nor extended loading of single-stranded flanking. Our protein-binding-ELISA experiments revealed specific and high-affinity binding of DDX5 to G4 structures regardless of whether the substrate is DNA or RNA. To elucidate the cellular functions of DDX5, we analyzed publicly available DDX5 ChIP-seq data and identified G-rich sequences in cancer cells as chromatin binding sites of DDX5. Moreover, our ChIP and Western Blot results showed that DDX5 is enriched at the MYC promoter and activates MYC transcription. Importantly, G-quadruplex interactive small molecules can inhibit the DDX5 interaction with the promoter MycG4 and DDX5-mediated transcriptional activation of the MYC gene. In addition, knock-down of DDX5 expression with DDX5-specific siRNA in cancer cells resulted in downregulation of MYC expression and sensitization to G4-interactive small molecules. In summary, we identified resolving DNA and RNA G-quadruplexes as novel function of DDX5. Our results establish that the MYC-transactivation by DDX5 is mediated through unfolding of the MYC promoter quadruplex and thereby establish a new molecular target to suppress MYC for cancer intervention. Citation Format: Guanhui Wu, Zheng Xing, Luying Chen, Elizabeth J. Tran, Danzhou Yang. DDX5 helicase resolves G-quadruplex and transactivates MYC expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3948.
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Drosopoulos, William C., Settapong T. Kosiyatrakul, and Carl L. Schildkraut. "BLM helicase facilitates telomere replication during leading strand synthesis of telomeres." Journal of Cell Biology 210, no. 2 (July 20, 2015): 191–208. http://dx.doi.org/10.1083/jcb.201410061.
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Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.
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Hellman, Lance M., Tyler J. Spear, Colton J. Koontz, Manana Melikishvili, and Michael G. Fried. "Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase." Nucleic Acids Research 42, no. 15 (July 30, 2014): 9781–91. http://dx.doi.org/10.1093/nar/gku659.
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Abstract O 6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.
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Tippana, Ramreddy, Helen Hwang, Patricia L. Opresko, Vilhelm A. Bohr, and Sua Myong. "Single-molecule imaging reveals a common mechanism shared by G-quadruplex–resolving helicases." Proceedings of the National Academy of Sciences 113, no. 30 (July 12, 2016): 8448–53. http://dx.doi.org/10.1073/pnas.1603724113.
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G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.
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van Kregten, Maartje, and Marcel Tijsterman. "The repair of G-quadruplex-induced DNA damage." Experimental Cell Research 329, no. 1 (November 2014): 178–83. http://dx.doi.org/10.1016/j.yexcr.2014.08.038.
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Heddi, Brahim, Vee Vee Cheong, Herry Martadinata, and Anh Tuân Phan. "Insights into G-quadruplex specific recognition by the DEAH-box helicase RHAU: Solution structure of a peptide–quadruplex complex." Proceedings of the National Academy of Sciences 112, no. 31 (July 20, 2015): 9608–13. http://dx.doi.org/10.1073/pnas.1422605112.
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Four-stranded nucleic acid structures called G-quadruplexes have been associated with important cellular processes, which should require G-quadruplex–protein interaction. However, the structural basis for specific G-quadruplex recognition by proteins has not been understood. The DEAH (Asp-Glu-Ala-His) box RNA helicase associated with AU-rich element (RHAU) (also named DHX36 or G4R1) specifically binds to and resolves parallel-stranded G-quadruplexes. Here we identified an 18-amino acid G-quadruplex-binding domain of RHAU and determined the structure of this peptide bound to a parallel DNA G-quadruplex. Our structure explains how RHAU specifically recognizes parallel G-quadruplexes. The peptide covers a terminal guanine base tetrad (G-tetrad), and clamps the G-quadruplex using three-anchor-point electrostatic interactions between three positively charged amino acids and negatively charged phosphate groups. This binding mode is strikingly similar to that of most ligands selected for specific G-quadruplex targeting. Binding to an exposed G-tetrad represents a simple and efficient way to specifically target G-quadruplex structures.
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Linke, Rebecca, Michaela Limmer, Stefan Juranek, Annkristin Heine, and Katrin Paeschke. "The Relevance of G-Quadruplexes for DNA Repair." International Journal of Molecular Sciences 22, no. 22 (November 22, 2021): 12599. http://dx.doi.org/10.3390/ijms222212599.
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DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.
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Sowers, Mark L., James W. Conrad, Bruce Chang-Gu, Ellie Cherryhomes, Linda C. Hackfeld, and Lawrence C. Sowers. "DNA Base Excision Repair Intermediates Influence Duplex–Quadruplex Equilibrium." Molecules 28, no. 3 (January 18, 2023): 970. http://dx.doi.org/10.3390/molecules28030970.
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Although genomic DNA is predominantly duplex under physiological conditions, particular sequence motifs can favor the formation of alternative secondary structures, including the G-quadruplex. These structures can exist within gene promoters, telomeric DNA, and regions of the genome frequently found altered in human cancers. DNA is also subject to hydrolytic and oxidative damage, and its local structure can influence the type of damage and its magnitude. Although the repair of endogenous DNA damage by the base excision repair (BER) pathway has been extensively studied in duplex DNA, substantially less is known about repair in non-duplex DNA structures. Therefore, we wanted to better understand the effect of DNA damage and repair on quadruplex structure. We first examined the effect of placing pyrimidine damage products uracil, 5-hydroxymethyluracil, the chemotherapy agent 5-fluorouracil, and an abasic site into the loop region of a 22-base telomeric repeat sequence known to form a G-quadruplex. Quadruplex formation was unaffected by these analogs. However, the activity of the BER enzymes were negatively impacted. Uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1) were inhibited, and apurinic/apyrimidinic endonuclease 1 (APE1) activity was completely blocked. Interestingly, when we performed studies placing DNA repair intermediates into the strand opposite the quadruplex, we found that they destabilized the duplex and promoted quadruplex formation. We propose that while duplex is the preferred configuration, there is kinetic conversion between duplex and quadruplex. This is supported by our studies using a quadruplex stabilizing molecule, pyridostatin, that is able to promote quadruplex formation starting from duplex DNA. Our results suggest how DNA damage and repair intermediates can alter duplex-quadruplex equilibrium.
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Gao, Jun, Zhaofeng Gao, Andrea A. Putnam, Alicia K. Byrd, Sarah L. Venus, John C. Marecki, Andrea D. Edwards, Haley M. Lowe, Eckhard Jankowsky, and Kevin D. Raney. "G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p." Chemical Communications 57, no. 60 (2021): 7445–48. http://dx.doi.org/10.1039/d1cc01479j.
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G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.
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Dallavalle, Sabrina, Salvatore Princiotto, Luce M. Mattio, Roberto Artali, Loana Musso, Anna Aviñó, Ramon Eritja, Claudio Pisano, Raimundo Gargallo, and Stefania Mazzini. "Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region." International Journal of Molecular Sciences 22, no. 16 (August 14, 2021): 8737. http://dx.doi.org/10.3390/ijms22168737.
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DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.
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Vinciguerra, Patrizia, Susana Godinho, Kalindi Parmar, David Pellman, and Alan D'Andrea. "Cytokinesis Failure in Fanconi Anemia Pathway Deficient Murine Hematopoietic Stem Cells." Blood 114, no. 22 (November 20, 2009): 495. http://dx.doi.org/10.1182/blood.v114.22.495.495.
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Abstract Abstract 495 Fanconi Anemia (FA) is a rare recessive chromosomal-instability disorder characterized by congenital malformations, a high predisposition to cancer, and progressive bone marrow failure. FA is genetically heterogeneous and, to date, thirteen FA genes have been identified (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N). The thirteen encoded FA proteins cooperate in a common DNA repair pathway active during the Synthesis (S) phase of the cell cycle. DNA damage detected during replication results in the monoubiquitination of two FA proteins, FANCD2 and FANCI, that translocate into chromatin-associated DNA repair foci where they colocalize with downstream components of the pathway. Partial colocalization with BLM, the RecQ helicase mutated in Bloom's syndrome, has also been described. How disruption of this pathway leads to bone marrow failure is a critical unanswered question. Interestingly, FA cells also have abnormalities that suggest a defect in mitosis, including micronuclei and multinucleation. The objectives of this study were to 1) investigate the role of the FA pathway in normal mitosis and 2) determine whether defects in this function underlie the bone marrow failure of FA patients. For this study, we used HeLa cells transiently or stably knocked down for FA genes, FA patient derived cell lines and hematopoietic stem cells from Fanconi mice models generated in our laboratory (Fancd2-/- and Fancg-/-). First, a polyclonal antibody was raised against FANCI and, together with an anti-FANCD2 antibody, used to investigate the localization of the FANCD2-I complex throughout the cell cycle by immunostaining. FANCI and FANCD2 colocalized to discrete foci on condensed chromosomes in a population of cells in Mitosis (M) phase, consistent with results of Chan et al. (Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol. 2009;11:753-760), Naim and Rosselli (The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol. 2009;11:761-768). These foci were dependent on an intact FA pathway, but did not localize at centromeres and did not increase when the spindle assembly checkpoint was challenged. By immunofluorescence, we showed an increase in the presence of Hoechst positive DNA bridges and PICH positive / BLM positive DNA bridges (Hoechst positive and negative) in anaphase and telophase of FA deficient cells compared to FA proficient cells. This increase of DNA bridges between separating sister chromatids in FA deficient cells correlated with an increase of multinucleated cells. Multinuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Furthermore, inhibition of apoptosis increased the number of binucleated cells, suggesting that cytokinesis failure led to apoptosis. Importantly, an increase in binucleated cells was also observed in the hematopoietic stem cells population from Fancd2-/- and Fancg-/- mice, compared to wild-type sibling mice, and this increase correlated with elevated apoptosis in those cells. Based on these new findings, we conclude that the Fanconi pathway is required for normal mitosis and hypothesize that apoptosis induced by cytokinesis failure of hematopoietic stem cells may cause the bone marrow failure commonly found in FA patients. Disclosures: No relevant conflicts of interest to declare.
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Wu, Guanhui, Zheng Xing, Elizabeth J. Tran, and Danzhou Yang. "DDX5 helicase resolves G-quadruplex and is involved in MYC gene transcriptional activation." Proceedings of the National Academy of Sciences 116, no. 41 (September 23, 2019): 20453–61. http://dx.doi.org/10.1073/pnas.1909047116.
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G-quadruplexes (G4) are noncanonical secondary structures formed in guanine-rich DNA and RNA sequences. MYC, one of the most critical oncogenes, forms a DNA G4 in its proximal promoter region (MycG4) that functions as a transcriptional silencer. However, MycG4 is highly stable in vitro and its regulatory role would require active unfolding. Here we report that DDX5, one of the founding members of the DEAD-box RNA helicase family, is extremely proficient at unfolding MycG4-DNA. Our results show that DDX5 is a highly active G4-resolvase that does not require a single-stranded overhang and that ATP hydrolysis is not directly coupled to G4-unfolding of DDX5. The chromatin binding sites of DDX5 are G-rich sequences. In cancer cells, DDX5 is enriched at the MYC promoter and activates MYC transcription. The DDX5 interaction with the MYC promoter and DDX5-mediated MYC activation is inhibited by G4-interactive small molecules. Our results uncover a function of DDX5 in resolving DNA and RNA G4s and suggest a molecular target to suppress MYC for cancer intervention.
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Paeschke, Katrin, John A. Capra, and Virginia A. Zakian. "DNA Replication through G-Quadruplex Motifs Is Promoted by the Saccharomyces cerevisiae Pif1 DNA Helicase." Cell 145, no. 5 (May 2011): 678–91. http://dx.doi.org/10.1016/j.cell.2011.04.015.
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Das, Tulika, Surasree Pal, and Agneyo Ganguly. "Human RecQ helicases in transcription-associated stress management: bridging the gap between DNA and RNA metabolism." Biological Chemistry 402, no. 5 (February 11, 2021): 617–36. http://dx.doi.org/10.1515/hsz-2020-0324.
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Abstract RecQ helicases are a highly conserved class of DNA helicases that play crucial role in almost all DNA metabolic processes including replication, repair and recombination. They are able to unwind a wide variety of complex intermediate DNA structures that may result from cellular DNA transactions and hence assist in maintaining genome integrity. Interestingly, a huge number of recent reports suggest that many of the RecQ family helicases are directly or indirectly involved in regulating transcription and gene expression. On one hand, they can remove complex structures like R-loops, G-quadruplexes or RNA:DNA hybrids formed at the intersection of transcription and replication. On the other hand, emerging evidence suggests that they can also regulate transcription by directly interacting with RNA polymerase or recruiting other protein factors that may regulate transcription. This review summarizes the up to date knowledge on the involvement of three human RecQ family proteins BLM, WRN and RECQL5 in transcription regulation and management of transcription associated stress.
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Götz, Silvia, Satyaprakash Pandey, Sabrina Bartsch, Stefan Juranek, and Katrin Paeschke. "A Novel G-Quadruplex Binding Protein in Yeast—Slx9." Molecules 24, no. 9 (May 7, 2019): 1774. http://dx.doi.org/10.3390/molecules24091774.
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G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.
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Zhou, Jia, Minmin Liu, Aaron M. Fleming, Cynthia J. Burrows, and Susan S. Wallace. "Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context." Journal of Biological Chemistry 288, no. 38 (August 7, 2013): 27263–72. http://dx.doi.org/10.1074/jbc.m113.479055.
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The telomeric DNA of vertebrates consists of d(TTAGGG)n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.
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Lin, Weiqiang, Shilpa Sampathi, Huifang Dai, Changwei Liu, Mian Zhou, Jenny Hu, Qin Huang, et al. "Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity." EMBO Journal 32, no. 10 (April 19, 2013): 1425–39. http://dx.doi.org/10.1038/emboj.2013.88.
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Maleki, Parastoo, Golam Mustafa, Prabesh Gyawali, Jagat B. Budhathoki, Yue Ma, Kazuo Nagasawa, and Hamza Balci. "Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase." Nucleic Acids Research 47, no. 20 (September 23, 2019): 10744–53. http://dx.doi.org/10.1093/nar/gkz803.
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Abstract G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.
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Stroik, Susanna, Kevin Kurtz, Kevin Lin, Sergey Karachenets, Chad L. Myers, Anja-Katrin Bielinsky, and Eric A. Hendrickson. "EXO1 resection at G-quadruplex structures facilitates resolution and replication." Nucleic Acids Research 48, no. 9 (March 31, 2020): 4960–75. http://dx.doi.org/10.1093/nar/gkaa199.
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Abstract G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.
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Amato, Jussara, Linda Cerofolini, Diego Brancaccio, Stefano Giuntini, Nunzia Iaccarino, Pasquale Zizza, Sara Iachettini, et al. "Insights into telomeric G-quadruplex DNA recognition by HMGB1 protein." Nucleic Acids Research 47, no. 18 (August 26, 2019): 9950–66. http://dx.doi.org/10.1093/nar/gkz727.
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Abstract HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
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Wu, Wenwen, Nana Rokutanda, Jun Takeuchi, Yongqiang Lai, Reo Maruyama, Yukiko Togashi, Hiroyuki Nishikawa, et al. "HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA." Cancer Research 78, no. 22 (October 2, 2018): 6371–85. http://dx.doi.org/10.1158/0008-5472.can-18-1877.
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Budhathoki, Jagat B., Parastoo Maleki, William A. Roy, Pavel Janscak, Jaya G. Yodh, and Hamza Balci. "A Comparative Study of G-Quadruplex Unfolding and DNA Reeling Activities of Human RECQ5 Helicase." Biophysical Journal 110, no. 12 (June 2016): 2585–96. http://dx.doi.org/10.1016/j.bpj.2016.05.016.
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Galati, Elena, Maria C. Bosio, Daniele Novarina, Matteo Chiara, Giulia M. Bernini, Alessandro M. Mozzarelli, Maria L. García-Rubio, et al. "VID22 counteracts G-quadruplex-induced genome instability." Nucleic Acids Research 49, no. 22 (December 6, 2021): 12785–804. http://dx.doi.org/10.1093/nar/gkab1156.
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Abstract Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.
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Gutierrez-Rodrigues, Fernanda, Sachiko Kajigaya, Xingmin Feng, Maria del Pilar Fernandez Ibanez, Marie J. Desierto, Keyvan Keyvanfar, Zejuan Li, et al. "Heterozygous RTEL1 variants in Patients with Bone Marrow Failure Associate with Telomere Dysfunction in the Absence of Telomere Shortening." Blood 128, no. 22 (December 2, 2016): 1044. http://dx.doi.org/10.1182/blood.v128.22.1044.1044.
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Abstract The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. Variability in penetrance and phenotype can complicate diagnosis, as patients with underlying genetic defects may present in adulthood and without characteristic physical anomalies. RTEL1 encodes a helicase crucial for telomere maintenance and DNA repair. The gene has two main transcripts in human cells: the 1300 amino acid isoform 3 and the 1219 amino acid isoform 1. RTEL1 isoform 3 contains a conserved C4C4-RING domain responsible for resolving the t-loop required for telomere replication. Using next-generation sequencing (NGS), RTEL1 germline variants with unknown clinical significance have been found in AA patients. Functional tests may elucidate RTEL1 variants' pathogenic role in telomere biology. Here, we describe RTEL1 heterozygous germline mutations in patients with BMF and investigate their impact in telomere maintenance. We screened 63 patients with a suggestive familial phenotype for germline mutations in peripheral blood cells using a targeted, 49 gene NGS panel. To investigate variants' impact in telomere functions, telomere length (TL) was measured by Southern blot (SB), t-circles were quantified by telomere circle assay, and single-stranded overhang was measured by non-denaturing SB. Eight patients carried novel heterozygous non-synonymous RTEL1 variants: four nucleotide changes were located in the RAD3 domain, six in the harmonin-like domain, and one in the RING domain. Clinical features and TL were heterogeneous (Table 1). The only RTEL1 variant predicted as pathogenic in silico was F1262L (c.3786 C>G) in patient 2; this mutation affects a highly conserved amino acid residue located in the RING domain, which is responsible for RTEL1 interaction with TRF2 at telomeres and t-loop unwinding. Patient 2 had very short telomeres, abnormal accumulation of t-circles, and eroded single-stranded telomeric overhangs in leukocytes, indicating a disrupted RTEL1 RING domain. To confirm observations made in clinical samples, 293T cells transfected with a plasmid carrying wild-type RTEL1-FLAG isoform 3 or its F1262L mutated version were assessed for TRF2 and FLAG co-localization in the nucleus. By confocal microscopy, wild-type RTEL1, but not mutant RTEL1 co-localized with TRF2. These findings strongly implicate RTEL1-F1262L as pathogenic, and thus the first autosomal dominant mutation in the RING domain in an AA patient. In patient 1, D743N variant in silico prediction was indeterminate, but telomeres were very short and there was a family history of typical telomeropathy (AA, liver cirrhosis, and pulmonary fibrosis) without any other suspicious germline mutations. The D743N variant is located close to the V745M variant that has been reported in a patient with dyskeratosis congenita. Increased amounts of t-circles and telomeric overhang attrition were observed in three other patients (#4, 5, and 7). While not specific for RTEL1 function, these results suggest telomere dysfunction, despite TLs in the normal range for patient 4 and 5. The RTEL1 P82L variant also appeared related to clonal evolution and leukemic progression observed in patient 5. For patients 3, 4, 6, 7, and 8, several mutations were observed in other genes concomitant to RTEL1, and a more complex genomic architecture may be the cause of patients' phenotype. A previously reported TERC variant, and a TERT variant of undetermined in silico prediction, could be pathogenic in patients 7 and 6, respectively. In these cases, RTEL1 variants may modulate disease, or represent only coincidental abnormalities. To our knowledge, this is the first report of heterozygous RTEL1 mutations in AA. We also describe a TL-independent association between RTEL1 haploinsufficiency and telomere dysfunction in humans. Haploinsufficiency of RTEL1 may disrupt DNA repair, destabilize the genome, and promote leukemogenesis by a mechanism different than typical accelerated telomere attrition associated with very short telomeres. T-circle quantification and overhang measurement may be better measures of telomere dysfunction in patients with RTEL1 variants than simple TL assessment. The combination of different functional tests was useful to the assessment of novel variants impact in telomere maintenance and DNA repair. Disclosures Fernandez Ibanez: GSK/Novartis: Research Funding. Desierto:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding.
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Obi, Ikenna, Matilda Rentoft, Vandana Singh, Jan Jamroskovic, Karam Chand, Erik Chorell, Fredrik Westerlund, and Nasim Sabouri. "Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication." Nucleic Acids Research 48, no. 19 (October 12, 2020): 10998–1015. http://dx.doi.org/10.1093/nar/gkaa820.
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Abstract G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.