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1
Gupta, Rigu, Sudha Sharma, Joshua A. Sommers, Mark K. Kenny, Sharon B. Cantor, and Robert M. Brosh. "FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein." Blood 110, no. 7 (October 1, 2007): 2390–98. http://dx.doi.org/10.1182/blood-2006-11-057273.
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The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability.
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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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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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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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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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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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Awate, Sanket, Joshua A. Sommers, Arindam Datta, Sumeet Nayak, Marina A. Bellani, Olivia Yang, Christopher A. Dunn, et al. "FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links." Nucleic Acids Research 48, no. 16 (August 14, 2020): 9161–80. http://dx.doi.org/10.1093/nar/gkaa660.
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Abstract FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.
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Pugh, Robert A., Masayoshi Honda, Haley Leesley, Alvin Thomas, Yuyen Lin, Mark J. Nilges, Isaac K. O. Cann, and Maria Spies. "The Iron-containing Domain Is Essential in Rad3 Helicases for Coupling of ATP Hydrolysis to DNA Translocation and for Targeting the Helicase to the Single-stranded DNA-Double-stranded DNA Junction." Journal of Biological Chemistry 283, no. 3 (November 20, 2007): 1732–43. http://dx.doi.org/10.1074/jbc.m707064200.
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Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-double-stranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.
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Mailand, Niels. "A DNA helicase remodeling proteins: How DNA-protein crosslink repair unfolds via FANCJ." Molecular Cell 83, no. 1 (January 2023): 3–5. http://dx.doi.org/10.1016/j.molcel.2022.12.009.
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Yaneva, Denitsa, Justin L. Sparks, Maximilian Donsbach, Shubo Zhao, Pedro Weickert, Rachel Bezalel-Buch, Julian Stingele, and Johannes C. Walter. "The FANCJ helicase unfolds DNA-protein crosslinks to promote their repair." Molecular Cell 83, no. 1 (January 2023): 43–56. http://dx.doi.org/10.1016/j.molcel.2022.12.005.
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Kumaraswamy, Easwari, and Ramin Shiekhattar. "Activation of BRCA1/BRCA2-Associated Helicase BACH1 Is Required for Timely Progression through S Phase." Molecular and Cellular Biology 27, no. 19 (July 30, 2007): 6733–41. http://dx.doi.org/10.1128/mcb.00961-07.
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ABSTRACT BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G1 phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability.
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Nath, Sarmi, and Ganesh Nagaraju. "FANCJ helicase promotes DNA end resection by facilitating CtIP recruitment to DNA double-strand breaks." PLOS Genetics 16, no. 4 (April 6, 2020): e1008701. http://dx.doi.org/10.1371/journal.pgen.1008701.
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Dohrn, Lisa, Daniela Salles, Simone Y. Siehler, Julia Kaufmann, and Lisa Wiesmüller. "BRCA1-mediated repression of mutagenic end-joining of DNA double-strand breaks requires complex formation with BACH1." Biochemical Journal 441, no. 3 (January 16, 2012): 919–28. http://dx.doi.org/10.1042/bj20110314.
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BACH1 (BRCA1-associated C-terminal helicase 1), the product of the BRIP1 {BRCA1 [breast cancer 1, early onset]-interacting protein C-terminal helicase 1; also known as FANCJ [FA-J (Fanconi anaemia group J) protein]} gene mutated in Fanconi anaemia patients from complementation group J, has been implicated in DNA repair and damage signalling. BACH1 exerts DNA helicase activities and physically interacts with BRCA1 and MLH1 (mutL homologue 1), which differentially control DNA DSB (double-strand break) repair processes. The present study shows that BACH1 plays a role in both HR (homologous recombination) and MMEJ (microhomology-mediated non-homologous end-joining) and reveals discrete mechanisms underlying modulation of these pathways. Our results indicate that BACH1 stimulates HR, which depends on the integrity of the helicase domain. Disruption of the BRCA1–BACH1 complex through mutation of BACH1 compromised errorfree NHEJ (non-homologous end-joining) and accelerated error-prone MMEJ. Conversely, molecular changes in BACH1 abrogating MLH1 binding interfered neither with HR nor with MMEJ. Importantly, MMEJ is a mutagenic DSB repair pathway, which is derepressed in hereditary breast and ovarian carcinomas. Since BRCA1 and BACH1 mutations targeting the BRCA1–BACH1 interaction have been associated with breast cancer susceptibility, the results of the present study thus provide evidence for a novel role of BACH1 in tumour suppression.
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Levitus, Marieke, Hans Joenje, and Johan P. de Winter. "The Fanconi Anemia Pathway of Genomic Maintenance." Analytical Cellular Pathology 28, no. 1-2 (January 1, 2006): 3–29. http://dx.doi.org/10.1155/2006/974975.
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Fanconi anemia (FA), a recessive syndrome with both autosomal and X-linked inheritance, features diverse clinical symptoms, such as progressive bone marrow failure, hypersensitivity to DNA cross-linking agents, chromosomal instability and susceptibility to cancer. At least 12 genetic subtypes have been described (FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA-I have been linked to a distinct gene. Most FA proteins form a complex that activates the FANCD2 protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream of this step. The FA proteins typically lack functional domains, except for FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross-linking agents, the FA proteins are thought to function in the repair of DNA interstrand cross-links, which block the progression of DNA replication forks. Here we present a hypothetical model, which not only describes the assembly of the FA pathway, but also positions this pathway in the broader context of DNA cross-link repair. Finally, the possible role for the FA pathway, in particular FANCF and FANCB, in the origin of sporadic cancer is discussed.
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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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Taniguchi, Toshiyasu, and Alan D. D'Andrea. "Molecular pathogenesis of Fanconi anemia: recent progress." Blood 107, no. 11 (June 1, 2006): 4223–33. http://dx.doi.org/10.1182/blood-2005-10-4240.
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AbstractA rare genetic disease, Fanconi anemia (FA), now attracts broader attention from cancer biologists and basic researchers in the DNA repair and ubiquitin biology fields as well as from hematologists. FA is a chromosome instability syndrome characterized by childhood-onset aplastic anemia, cancer or leukemia susceptibility, and cellular hypersensitivity to DNAcrosslinking agents. Identification of 11 genes for FA has led to progress in the molecular understanding of this disease. FA proteins, including a ubiquitin ligase (FANCL), a monoubiquitinated protein (FANCD2), a helicase (FANCJ/BACH1/BRIP1), and a breast/ovarian cancer susceptibility protein (FANCD1/BRCA2), appear to cooperate in a pathway leading to the recognition and repair of damaged DNA. Molecular interactions among FA proteins and responsible proteins for other chromosome instability syndromes (BLM, NBS1, MRE11, ATM, and ATR) have also been found. Furthermore, inactivation of FA genes has been observed in a wide variety of human cancers in the general population. These findings have broad implications for predicting the sensitivity and resistance of tumors to widely used anticancer DNA crosslinking agents (cisplatin, mitomycin C, and melphalan). Here, we summarize recent progress in the molecular biology of FA and discuss roles of the FA proteins in DNA repair and cancer biology.
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Calvo, Jennifer A., Briana Fritchman, Desiree Hernandez, Nicole S. Persky, Cory M. Johannessen, Federica Piccioni, Brian A. Kelch, and Sharon B. Cantor. "Comprehensive Mutational Analysis of the BRCA1-Associated DNA Helicase and Tumor-Suppressor FANCJ/BACH1/BRIP1." Molecular Cancer Research 19, no. 6 (February 22, 2021): 1015–25. http://dx.doi.org/10.1158/1541-7786.mcr-20-0828.
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Dorn, Annika, Laura Feller, Dominique Castri, Sarah Röhrig, Janina Enderle, Natalie J. Herrmann, Astrid Block-Schmidt, Oliver Trapp, Laura Köhler, and Holger Puchta. "An Arabidopsis FANCJ helicase homologue is required for DNA crosslink repair and rDNA repeat stability." PLOS Genetics 15, no. 5 (May 23, 2019): e1008174. http://dx.doi.org/10.1371/journal.pgen.1008174.
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Wu, Yuliang, Joshua A. Sommers, Jason A. Loiland, Hiroyuki Kitao, Jochen Kuper, Caroline Kisker, and Robert M. Brosh. "The Q Motif of Fanconi Anemia Group J Protein (FANCJ) DNA Helicase Regulates Its Dimerization, DNA Binding, and DNA Repair Function." Journal of Biological Chemistry 287, no. 26 (May 10, 2012): 21699–716. http://dx.doi.org/10.1074/jbc.m112.351338.
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Wu, Yuliang, and Robert Brosh Jr. "FANCJ Helicase Operates in the Fanconi Anemia DNA Repair Pathway and the Response to Replicational Stress." Current Molecular Medicine 9, no. 4 (May 1, 2009): 470–82. http://dx.doi.org/10.2174/156652409788167159.
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London, Timothy B. C., Louise J. Barber, Georgina Mosedale, Gavin P. Kelly, Shankar Balasubramanian, Ian D. Hickson, Simon J. Boulton, and Kevin Hiom. "FANCJ Is a Structure-specific DNA Helicase Associated with the Maintenance of Genomic G/C Tracts." Journal of Biological Chemistry 283, no. 52 (October 31, 2008): 36132–39. http://dx.doi.org/10.1074/jbc.m808152200.
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Mani, Chinnadurai, Ganesh Acharya, Sudhir Kshirsagar, Murali Vijayan, Hafiz Khan, P. Hemachandra Reddy, and Komaraiah Palle. "A Novel Role for BRIP1/FANCJ in Neuronal Cells Health and in Resolving Oxidative Stress-Induced DNA Lesions." Journal of Alzheimer's Disease 85, no. 1 (January 4, 2022): 207–21. http://dx.doi.org/10.3233/jad-215305.
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Background: DNA damage accumulation and mitochondrial abnormalities are elevated in neurons during aging and may contribute to neurodegenerative pathologic conditions such as Alzheimer’s disease. BRCA1 interacting protein 1 or BRIP1 is a 5’ to 3’ DNA helicase that catalyzes many abnormal DNA structures during DNA replication, gene transcription, and recombination, and contribute to genomic integrity. Objective: BRIP1 functions were reasonably well studied in DNA repair; however, there is limited data on its role and regulation during aging and neurodegenerative diseases. Methods: We used immunohistochemistry, western blot, and qRT-PCR assays to analyze the expression of BRIP1. Immunofluorescence studies were performed to study the formation of R-loops, reactive oxygen species (ROS) generation, and mitochondrial morphology. Flow cytometry and transmission electron microscopy were used to evaluate mitochondrial ROS and mitochondrial structures, respectively. Oxygen consumption rate was measured using Seahorse, and the Presto Blue™ assays were used to evaluate cell viability. Results: Our results demonstrate the expression of BRIP1 in mouse and human brain tissues and in neuronal cell lines. BRIP1 levels were elevated in the hippocampal regions of the brains, specifically in the dentate gyrus. BRIP1 downregulation in neuronal cells caused increased R-loop formation basally and in response to H2O2 treatment. Furthermore, BRIP1 deficient cells exhibited elevated levels of excitotoxicity induced by L-Glutamic acid exposure as evidenced by (mitochondrial) ROS levels, deteriorated mitochondrial health, and cell death compared to BRIP1 proficient neuronal cells. Conclusion: Overall, our results indicate an important role for BRIP1 in maintaining neuronal cell health and homeostasis by suppressing cellular oxidative stress.
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Stone, Stacie, Alexandra Sobeck, Igor Landais, Alexis LaChapelle, and Maureen E. Hoatlin. "A Cell-Free Assay To Screen for Compounds That Modulate the Fanconi/BRCA Pathway." Blood 108, no. 11 (November 16, 2006): 4351. http://dx.doi.org/10.1182/blood.v108.11.4351.4351.
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Abstract Fanconi anemia is a multi-gene cancer susceptibility and bone marrow failure syndrome. In the current model, at least eight proteins (FANCA, -B-C,-E, -F, -G, -L, -M) are part of a nuclear complex that is required for the S phase and DNA-damage dependent monoubiquitination of FANCD2. This event is thought to functionally link the FA complex proteins to major breast cancer susceptibility proteins BRCA1, BRCA2 (FANCD1), and the BRCA1-associated helicase Brip1(FANCJ). An understanding of the function of the FA protein network is incomplete not only because some FA proteins are still unidentified, but also because the functions of individual proteins may be interdependent and are difficult to assess out of context with the entire FA network. We recently developed a cell-free system to evaluate the function of the Fanconi/BRCA pathway proteins in an S phase context in Xenopus egg extracts (Sobeck, et al. 2006). Egg extracts are naturally cell-cycle synchronized and mimic the complex interplay of proteins that support cellular DNA replication and regulated DNA damage checkpoint activation. Intricate protein interactions can be assayed in egg extracts, even without knowing each of the components if there is a measurable endpoint. We tested the hypothesis that the mobility shift of FANCD2 could be used as an endpoint in cell-free assays to determine FA pathway function. We found that an antibody specific for the Xenopus FANCD2 protein detected a single band of the expected size in western blots of proteins separated by SDS-PAGE from unstimulated egg extracts. Addition of DNA substrates to extracts resulted in the appearance of a slower mobility form of FANCD2, consistent with the monoubiquitinated FANCD2-L isoform observed in human cells following DNA damage. We measured inhibition or stimulation of xFANCD2-L in the presence of a series of candidate compounds. We found compounds that inhibit FANCD2-L, including curcumin, which was also identified in a cell-based assay as an inhibitor of FANCD2-L (Chirnomas, et al., 2006). Thus, this cell-free assay successfully mirrors the outcome obtained with a small molecule inhibitor of the FA/BRCA pathway in cell-based assays. This new approach is an improvement relative to cell-based screens because the extracts are fully synchronized, which maximizes the sensitivity of detection of S-phase events. Moreover, cell-free screens are rapid, inexpensive and well suited for semi- or high-throughput methods to identify small molecules that modulate the FA/BRCA DNA-damage response pathway.
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Suhasini, Avvaru N., Joshua A. Sommers, Aaron C. Mason, Oleg N. Voloshin, R. Daniel Camerini-Otero, Marc S. Wold, and Robert M. Brosh. "FANCJ Helicase Uniquely Senses Oxidative Base Damage in Either Strand of Duplex DNA and Is Stimulated by Replication Protein A to Unwind the Damaged DNA Substrate in a Strand-specific Manner." Journal of Biological Chemistry 284, no. 27 (May 5, 2009): 18458–70. http://dx.doi.org/10.1074/jbc.m109.012229.
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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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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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Enderle, Janina, Annika Dorn, and Holger Puchta. "DNA- and DNA-Protein-Crosslink Repair in Plants." International Journal of Molecular Sciences 20, no. 17 (September 3, 2019): 4304. http://dx.doi.org/10.3390/ijms20174304.
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DNA-crosslinks are one of the most severe types of DNA lesions. Crosslinks (CLs) can be subdivided into DNA-intrastrand CLs, DNA-interstrand CLs (ICLs) and DNA-protein crosslinks (DPCs), and arise by various exogenous and endogenous sources. If left unrepaired before the cell enters S-phase, ICLs and DPCs pose a major threat to genomic integrity by blocking replication. In order to prevent the collapse of replication forks and impairment of cell division, complex repair pathways have emerged. In mammals, ICLs are repaired by the so-called Fanconi anemia (FA) pathway, which includes 22 different FANC genes, while in plants only a few of these genes are conserved. In this context, two pathways of ICL repair have been defined, each requiring the interaction of a helicase (FANCJB/RTEL1) and a nuclease (FAN1/MUS81). Moreover, homologous recombination (HR) as well as postreplicative repair factors are also involved. Although DPCs possess a comparable toxic potential to cells, it has only recently been shown that at least three parallel pathways for DPC repair exist in plants, defined by the protease WSS1A, the endonuclease MUS81 and tyrosyl-DNA phosphodiesterase 1 (TDP1). The importance of crosslink repair processes are highlighted by the fact that deficiencies in the respective pathways are associated with diverse hereditary disorders.
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Thomas, Adam, Julie Cox, Kelly B. Wolfe, Carrie Hui Mingalone, Haleigh R. Yaspan, and Mitch McVey. "Division of Labor by the HELQ, BLM, and FANCM Helicases during Homologous Recombination Repair in Drosophila melanogaster." Genes 13, no. 3 (March 8, 2022): 474. http://dx.doi.org/10.3390/genes13030474.
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Repair of DNA double-strand breaks by homologous recombination (HR) requires a carefully orchestrated sequence of events involving many proteins. One type of HR, synthesis-dependent strand annealing (SDSA), proceeds via the formation of a displacement loop (D-loop) when RAD51-coated single-stranded DNA invades a homologous template. The 3′ end of the single-stranded DNA is extended by DNA synthesis. In SDSA, the D-loop is then disassembled prior to strand annealing. While many helicases can unwind D-loops in vitro, how their action is choreographed in vivo remains to be determined. To clarify the roles of various DNA helicases during SDSA, we used a double-strand gap repair assay to study the outcomes of homologous recombination repair in Drosophila melanogaster lacking the BLM, HELQ, and FANCM helicases. We found that the absence of any of these three helicases impairs gap repair. In addition, flies lacking both BLM and HELQ or HELQ and FANCM had more severe SDSA defects than the corresponding single mutants. In the absence of BLM, a large percentage of repair events were accompanied by flanking deletions. Strikingly, these deletions were mostly abolished in the blm helq and blm fancm double mutants. Our results suggest that the BLM, HELQ, and FANCM helicases play distinct roles during SDSA, with HELQ and FANCM acting early to promote the formation of recombination intermediates that are then processed by BLM to prevent repair by deletion-prone mechanisms.
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Whitby, Matthew C. "The FANCM family of DNA helicases/translocases." DNA Repair 9, no. 3 (March 2010): 224–36. http://dx.doi.org/10.1016/j.dnarep.2009.12.012.
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García-Luis, Jonay, and Félix Machín. "Fanconi Anaemia-Like Mph1 Helicase Backs up Rad54 and Rad5 to Circumvent Replication Stress-Driven Chromosome Bridges." Genes 9, no. 11 (November 17, 2018): 558. http://dx.doi.org/10.3390/genes9110558.
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Homologous recombination (HR) is a preferred mechanism to deal with DNA replication impairments. However, HR synapsis gives rise to joint molecules (JMs) between the nascent sister chromatids, challenging chromosome segregation in anaphase. Joint molecules are resolved by the actions of several structure-selective endonucleases (SSEs), helicases and topoisomerases. Previously, we showed that yeast double mutants for the Mus81-Mms4 and Yen1 SSEs lead to anaphase bridges (ABs) after replication stress. Here, we have studied the role of the Mph1 helicase in preventing these anaphase aberrations. Mph1, the yeast ortholog of Fanconi anaemia protein M (FANCM), is involved in the removal of the D-loop, the first JM to arise in canonical HR. Surprisingly, the absence of Mph1 alone did not increase ABs; rather, it blocked cells in G2. Interestingly, in the search for genetic interactions with functionally related helicases and translocases, we found additive effects on the G2 block and post-G2 aberrations between mph1Δ and knockout mutants for Srs2, Rad54 and Rad5. Based on these interactions, we suggest that Mph1 acts coordinately with these helicases in the non-canonical HR-driven fork regression mechanism to bypass stalled replication forks.
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Lansdorp, Peter, and Niek van Wietmarschen. "Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo." Genes 10, no. 11 (October 31, 2019): 870. http://dx.doi.org/10.3390/genes10110870.
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Guanine quadruplex (G4) structures are among the most stable secondary DNA structures that can form in vitro, and evidence for their existence in vivo has been steadily accumulating. Originally described mainly for their deleterious effects on genome stability, more recent research has focused on (potential) functions of G4 structures in telomere maintenance, gene expression, and other cellular processes. The combined research on G4 structures has revealed that properly regulating G4 DNA structures in cells is important to prevent genome instability and disruption of normal cell function. In this short review we provide some background and historical context of our work resulting in the identification of FANCJ, RTEL1 and BLM as helicases that act on G4 structures in vivo. Taken together these studies highlight important roles of different G4 DNA structures and specific G4 helicases at selected genomic locations and telomeres in regulating gene expression and maintaining genome stability.
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Séguéla-Arnaud, Mathilde, Wayne Crismani, Cécile Larchevêque, Julien Mazel, Nicole Froger, Sandrine Choinard, Afef Lemhemdi, et al. "Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM." Proceedings of the National Academy of Sciences 112, no. 15 (March 30, 2015): 4713–18. http://dx.doi.org/10.1073/pnas.1423107112.
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Meiotic crossovers (COs) have two important roles, shuffling genetic information and ensuring proper chromosome segregation. Despite their importance and a large excess of precursors (i.e., DNA double-strand breaks, DSBs), the number of COs is tightly regulated, typically one to three per chromosome pair. The mechanisms ensuring that most DSBs are repaired as non-COs and the evolutionary forces imposing this constraint are poorly understood. Here we identified Topoisomerase3α (TOP3α) and the RECQ4 helicases—the Arabidopsis slow growth suppressor 1 (Sgs1)/Bloom syndrome protein (BLM) homologs—as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, in addition to its role in DNA repair, this topoisomerase antagonizes CO formation. Further, we found that RECQ4A and RECQ4B constitute the strongest meiotic anti-CO activity identified to date, their concomitant depletion leading to a sixfold increase in CO frequency. In both top3α and recq4ab mutants, DSB number is unaffected, and extra COs arise from a normally minor pathway. Finally, both TOP3α and RECQ4A/B act independently of the previously identified anti-CO Fanconi anemia of complementation group M (FANCM) helicase. This finding shows that several parallel pathways actively limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is restricted not because of mechanical constraints but likely because of the long-term costs of recombination. Furthermore, this work demonstrates how manipulating a few genes holds great promise for increasing recombination frequency in plant-breeding programs.
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Rudolf, Jana, Vasso Makrantoni, W. John Ingledew, Michael J. R. Stark, and Malcolm F. White. "The DNA Repair Helicases XPD and FancJ Have Essential Iron-Sulfur Domains." Molecular Cell 23, no. 6 (September 2006): 801–8. http://dx.doi.org/10.1016/j.molcel.2006.07.019.
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Alix-Panabières, Catherine, Laure Cayrefourcq, Thibault Mazard, Thierry Maudelonde, Eric Assenat, and Said Assou. "Molecular Portrait of Metastasis-Competent Circulating Tumor Cells in Colon Cancer Reveals the Crucial Role of Genes Regulating Energy Metabolism and DNA Repair." Clinical Chemistry 63, no. 3 (March 1, 2017): 700–713. http://dx.doi.org/10.1373/clinchem.2016.263582.
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AbstractBACKGROUNDUnraveling the molecular mechanisms that regulate the biology of metastasis-competent circulating tumor cells (CTCs) is urgently needed to understand metastasis formation and tumor relapse. Our group previously established the first cell line (CTC-MCC-41) derived from metastasis-competent CTCs of a patient with colon cancer.METHODSIn this study, we analyzed the transcriptome of CTC-MCC-41 cells using Human Genome U133 Plus 2.0 microarrays with the aim of unraveling the molecular basis of their special features (stem cell properties and ability to initiate and support metastasis formation).RESULTSComparison of the transcriptome data of metastasis-competent CTC-MCC-41 cells and of HT-29 cells (derived from a primary colon cancer) highlights the differential expression of genes that regulate energy metabolism [peroxisome proliferator-activated receptor γ coactivator 1A (PPARGC1A), peroxisome proliferator-activated receptor γ coactivator 1B (PPARGC1B), fatty acid binding protein 1 (FABP1), aldehyde dehydrogenase 3 family member A1 (ALDH3A1)], DNA repair [BRCA1 interacting protein C-terminal helicase 1 (BRIP1), Fanconi anemia complementation group B (FANCB), Fanconi anemia complementation group M (FANCM)], and stemness [glutaminase 2 (GLS2), cystathionine-beta-synthase (CBS), and cystathionine gamma-lyase (CTH)]. The differential expression of 20 genes was validated by quantitative reverse transcription PCR.CONCLUSIONSThis study gives a comprehensive outlook on the molecular events involved in colon cancer progression and provides potential CTC biomarkers that may help develop new therapies to specifically target CTCs with stem cell properties that cause metastases and tumor relapse in patients with colon cancer.
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Parmar, Kalindi, Patrizia Vinciguerra, Susana Godinho, Abigail Hamilton, David Pellman, and Alan D. D'Andrea. "Cytokinesis Failure In Fanconi Anemia Pathway Deficient Hematopoietic Cells." Blood 116, no. 21 (November 19, 2010): 878. http://dx.doi.org/10.1182/blood.v116.21.878.878.
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Abstract Abstract 878 Fanconi Anemia (FA) is a human genomic instability disorder characterized by progressive bone marrow failure, congenital abnormalities and high predisposition to cancer. Bone marrow failure in FA children is attributed partly to the excessive apoptosis and subsequent failure of the hematopoietic stem cell compartment. Understanding the mechanisms of bone marrow failure may allow better diagnosis and treatment for FA and other aplastic anemia patients. There are fourteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O). The FA pathway, regulated by these FA gene products, mediates DNA repair and promotes normal cellular resistance to DNA crosslinking agents. Recent studies suggest that besides maintaining genomic stability, the FA pathway may also play a role in mitosis since FANCD2 and FANCI, the two key FA proteins, are localized to the extremities of ultra-fine DNA bridges (UFBs) linking sister chromatids during cell division (Chan et al, Nat Cell Biol, 11:753-760, 2009; Naim and Rosselli, Nat Cell Biol, 11:761-768, 2009). Whether FA proteins play a direct role in cell division is still unclear. To dissect the mechanisms of bone marrow failure in FA, we have investigated the requirement of FA pathway during mitosis. Initially, we investigated the number of DNA bridges occurring during mitosis in FA-deficient and proficient cells by immunofluorescence and Hoechst staining. FA-deficient patient cell lines (FANCG-deficient and FANCD1/BRCA2-deficient cells) as well as Hela cells with shRNA-mediated knockdown of the FA pathway, displayed an increase in UFBs compared to the FA proficient cells during mitosis. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein, FANCM, was recruited to the bridges at a later stage. Since the DNA bridges occluding the cleavage furrow potentially induce cytokinesis failure, we assessed FA-deficient cells for multinucleation. The increased number of DNA bridges correlated with a higher rate of binucleated cells in FA deficient Hela cell lines and FA patient-derived fibroblast cells. Moreover, an increase in binucleated cells was also detectable in FA-deficient primary murine bone marrow hematopoietic stem cells (Fancd2-/- cells and Fancg-/- cells) compared to the wild-type cells undergoing proliferation and in FA patient-derived bone marrow stroma cells compared to the stroma cells from normal human bone marrow. Interestingly, the increase in binucleated cells in FA-deficient murine hematopoietic stem cells correlated with the increase in apoptotic cells. Binuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Therefore, we investigated whether the FA-deficient cells are sensitive to the cytokinesis inhibitors. FA-deficient murine bone marrow lineage negative cells (Fancd2-/- cells) or FA human fibroblast cells were exposed to VX-680 (an inhibitor of Aurora kinases regulating cytokinesis) in culture for 72 hrs and cell survival was assessed. VX-680 caused increased toxicity (reduced cell viability and increased apoptosis) on FA-deficient cells in comparison to the wild-type cells. Enhanced inhibition of clonogenic growth of murine FA-deficient bone marrow cells (Fancd2-/- cells) compared to the wild-type cells was also observed by exposure to VX-680. These data indicated that FA pathway-deficient hematopoietic cells are hypersensitive to cytokinesis inhibitors. Collectively, our results underscore the importance of the FA pathway in mitosis and suggest that the cytokinesis failure observed in FA deficient hematopoietic cells could contribute to bone marrow failure in Fanconi anemia patients. Disclosures: No relevant conflicts of interest to declare.
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Stoepker, Chantal, Atiq Faramarz, Martin A. Rooimans, Saskia E. van Mil, Jesper A. Balk, Eunike Velleuer, Najim Ameziane, Hein te Riele, and Johan P. de Winter. "DNA helicases FANCM and DDX11 are determinants of PARP inhibitor sensitivity." DNA Repair 26 (February 2015): 54–64. http://dx.doi.org/10.1016/j.dnarep.2014.12.003.
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Liang, Fengshan, Arvindhan Nagarajan, Manoj M. Pillai, Patrick Sung, and Gary M. Kupfer. "Fanci-FANCD2 Promotes Genome Stability and DNA Repair By Down-Regulating BLM Helicase Activity." Blood 138, Supplement 1 (November 5, 2021): 1113. http://dx.doi.org/10.1182/blood-2021-152218.
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Abstract Background: Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair. Methods: Highly purified proteins were used to investigate how ID2 affects helicase and DNA end resection activity of the BLM complex. HeLa, FANCD2-deficient, and FANCD2 corrected fibroblast cell lines were used to examine pRPA2 and RAD51 foci formation. We also used DNA fiber assay to detect end resection and isolation of proteins on nascent DNA (iPOND) assay to examine the RAD51 recruitment on replication fork. Results: A somatic 6 amino acid deletion (p819-824) in FANCD2 was identified in a head and neck tumor. FA-D2 mutant cells expressing the mutant cDNA demonstrated defects in FANCD2 mono-ubiquitination and DNA damage hypersensitivity. A FANCD2-L822A mutant with defective BLM binding was identified (Figure A, B). We found that Bloom helicase and its DNA end resection activity within BLM-DNA2-RPA were negatively regulated by the heterodimer ID2 (Figure C, D). Both DNA and BLM binding of the ID2 are required for the inhibitory function. The premature DNA end resection and HU sensitivity in FANCD2 deficient and mutant cells are rescued by BLM knockdown. By iPOND assay, we discovered that FANCD2 antagonizes BLM to promote RAD51 recruitment on HU-stalled replication fork. Conclusions: Our study suggests that the DNA end resection activity of BLM-DNA2 is tightly regulated by FANCD2 to ensure that the nuclease DNA2 normally resects the DNA intermediate needed for efficient DNA repair and RAD51 recruitment to protect replication forks. Our findings highlight that ID2-BLM interaction functions in DNA damage repair to maintain genome stability. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Romero, N. E., S. W. Matson, and J. Sekelsky. "Biochemical Activities and Genetic Functions of the Drosophila melanogaster Fancm Helicase in DNA Repair." Genetics 204, no. 2 (July 27, 2016): 531–41. http://dx.doi.org/10.1534/genetics.116.192534.
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Stivison, Elizabeth A., Kati J. Young, and Lorraine S. Symington. "Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres." Nucleic Acids Research 48, no. 22 (December 2, 2020): 12697–710. http://dx.doi.org/10.1093/nar/gkaa1081.
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Abstract Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.
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Zhu, Longfei, Nadia Fernández-Jiménez, Maja Szymanska-Lejman, Alexandre Pelé, Charles J. Underwood, Heïdi Serra, Christophe Lambing, et al. "Natural variation identifies SNI1, the SMC5/6 component, as a modifier of meiotic crossover in Arabidopsis." Proceedings of the National Academy of Sciences 118, no. 33 (August 12, 2021): e2021970118. http://dx.doi.org/10.1073/pnas.2021970118.
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The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1. The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.
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Bagby, Grover C., Shannon McWeeney, Jane Yates, Byung Park, R. Keaney Rathbun, Daniela Pilonetto, Richard Harris, et al. "Comparative Functional Genomic Analysis of Myelodysplasia (MDS) in Fanconi Anemia (FA)." Blood 108, no. 11 (November 16, 2006): 2636. http://dx.doi.org/10.1182/blood.v108.11.2636.2636.
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Abstract FA stem cells are apoptotic, genetically unstable, and hypersensitive to a variety of apoptosis-inducing extracellular biological cues. To explain the high relative risk of MDS in FA patients, we propose that the combined influences of genetic instability and high-level ground-state stem cell apoptosis represent de-facto selective pressures that favor the emergence of stem cell clones that have become more fit as a result of somatic mutations. Indeed, we have previously reported that FA progenitor cells (FA) are hypersensitive to apoptotic proteins and that those derived from cytogenetically abnormal somatically mutated FA clones (FA-MDS) are resistant. To validate this adaptive model of clonal evolution on a genome-wide basis, we tested the hypothesis that the transcriptomal state of clonally evolved FA marrow cells (FA-MDS) is more closely related to that of normal (N) bone marrow cells than are marrow cells from non-clonal FA cells (FA). We compared transcriptomes of three groups of human and murine low density marrow cells; N, FA, and FA-MDS. RNA was obtained from 41 human samples: N=11, FA=21, FA-MDS=9. We also utilized marrow cells from wild type (N, n= 3), non clonal hypoplastic Fancc−/−//Fancg−/− double knockout (FA, n= 3), and clonal Fancc−/−//Fancg−/− (FA-MDS, n = 3) mice. Complementary RNA fragments were labeled for use as targets of the probes on Affymetrix chips (U133A for human and MOE 430 2.0 for murine samples). MAS 5.0 was utilized to process images, quantify signals, adjust background, and to scale the data. Pattern analysis, hierarchical clustering, and principle component analysis were performed using GeneSifter and SAS. Using either murine or human samples, unsupervised hierarchical clustering and multidimensional scaling demonstrated that expression patterns of N and FA samples were most dissimilar. However, the vectors of FA-MDS samples referred to points in expression space more closely linked to N samples. These observations support the notion that cytogenetically marked clones evolve adaptively from initially pro-apoptotic stem cells. To find differentially expressed orthologs of interest, we sought genes that fell into 2 specific expression patterns in each of which FA-MDS and N cells were not different but in which expression in FA cells was: suppressed (transcripts = 2084 human, 475 murine, 29 shared between murine and human) or increased (transcripts = 2222 human, 352 murine, 18 shared). Represented in the gene list suppressed in FA (and normally expressed in FA-MDS) were genes encoding proteins involved in: responses to factor-deprivation, signal transduction (beta-catenin, integrin, GTPase, phosphatidyl inositol, 14–3-3, and STAT), control of mitosis and mitotic spindle checkpoint, RNA polymerase II function, DNA-binding helicases, and ribosomal RNA processing and synthesis. In conclusion, using a comparative expression genomic approach we have validated the adaptive model of clonal evolution in FA and have significantly reduced the number of candidate genes involved either directly or indirectly in the molecular pathogenesis of MDS in Fanconi anemia.
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Jalas, Chaim, Anastasia Fedick, Bari J. Ballew, Blanche P. Alter, Neelam Giri, Simon Boulton, Kenneth Offit, John Petrini, Nathan Treff, and Sharon A. Savage. "Higher Than Expected Carrier Frequency Of The Dyskeratosis Congenita RTEL1 p.Arg1264His recessive Founder In Individuals Of Ashkenazi Jewish Ancestry." Blood 122, no. 21 (November 15, 2013): 1228. http://dx.doi.org/10.1182/blood.v122.21.1228.1228.
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Abstract Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure syndrome (IBMFS) in which germline mutations in telomere biology genes account for approximately 70% of known families. DC is clinically diagnosed by the presence of the triad of nail dysplasia, lacy skin pigmentation, and oral leukoplakia. However, not all patients have the triad and multiple other medical problems may include, stenosis of the esophagus, urethra and/or lacrimal ducts, avascular necrosis of the hips or shoulders, developmental delay, head and neck squamous cell cancer, and/or leukemia. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients have features of DC but also have microcephaly, cerebellar hypoplasia, and intrauterine growth retardation, and may present with severe immunodeficiency and enteropathy. Telomere lengths (in blood leukocyte subsets analyzed by flow FISH) less than the 1st percentile for age are diagnostic of any form of DC, including HH. We identified a germline autosomal recessive (AR) mutation (p.Arg1264His) in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The minor allele frequency of this variant is ∼0.0001 in public databases of 9600 individuals. The affected individuals in these families are homozygous for this mutation, which affects three isoforms of RTEL1. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In both families, each parent was a healthy, heterozygous carrier of one mutant allele. Haplotypes were reconstructed from twelve common SNPs based on allele sharing in the unaffected siblings and parents. No recombinants were seen in either family and the segregating risk haplotype was identical in affected individuals from both families. Thus, p.Arg1264His is carried on a common haplotype, likely from a common AJ founder. We determined the carrier frequency of the p.Arg1264His mutation, as well as three other mutations, p.Gly763Val, p.Met516Ile and p.Arg998Ter, which were recently reported and possibly found in individuals of AJ ancestry. DNA was derived from 1,048 self-described AJ individuals enrolled in the Dor Yeshorim program. Consent form information included that patient material would be used for clinical testing and that excess material would be de-identified and used for research purposes. The mutations were genotyped by TaqMan assays and heterozygous carrier samples were confirmed by Sanger sequencing with stringent quality control. No individuals in this study carried the p.Gly763Val or p.Arg998Ter minor alleles. Two individuals (0.19%) were carriers of the p.Met516Ile mutation. Notably, 1% (10 of 1,032) of AJ individuals in this study were carriers of the p.Arg1264His mutation in RTEL1. This carrier frequency of 1 in 100 is similar to that of the FANCC AJ mutation and many other FA founder populations, such as FANCA in South African Afrikaners, Spanish Gypsies, Brazilians, Tunisians, and Moroccans, as well as FANCG in Sub-Saharan Blacks, and BRCA2 (FANCD1) in the US general population. A carrier frequency of 1 in 100 is similar to that of genetic disorders found in the AJ population recommended for screening by the American College of Medical Genetics. Based on this, we suggest that genetic counseling and RTEL1 p.Arg1264His carrier screening for the HH variant of DC be offered to individuals of AJ ancestry. Disclosures: No relevant conflicts of interest to declare.
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Yeom, Gyuho, Jinwoo Kim, and Chin-Ju Park. "Investigation of the core binding regions of human Werner syndrome and Fanconi anemia group J helicases on replication protein A." Scientific Reports 9, no. 1 (September 30, 2019). http://dx.doi.org/10.1038/s41598-019-50502-8.
Full textAbstract:
Abstract Werner syndrome protein (WRN) and Fanconi anemia group J protein (FANCJ) are human DNA helicases that contribute to genome maintenance. They interact with replication protein A (RPA), and these interactions dramatically enhance the unwinding activities of both helicases. Even though the interplay between these helicases and RPA is particularly important in the chemoresistance pathway of cancer cells, the precise binding regions, interfaces, and properties have not yet been characterized. Here we present systematic NMR analyses and fluorescence polarization anisotropy assays of both helicase-RPA interactions for defining core binding regions and binding affinities. Our results showed that two acidic repeats of human WRN bind to RPA70N and RPA70A. For FANCJ, the acidic-rich sequence in the C-terminal domain is the binding region for RPA70N. Our results suggest that each helicase interaction has unique features, although they both fit an acidic peptide into a basic cleft for RPA binding. Our findings shed light on the protein interactions involved in overcoming the DNA-damaging agents employed in the treatment of cancer and thus potentially provide insight into enhancing the efficacy of cancer therapy.
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K, Jagadeesh Chandra Bose, Bishwajit Singh Kapoor, Kamal Mandal, Shubhrima Ghosh, Raveendra B. Mokhamatam, Sunil K. Manna, and Sudit S. Mukhopadhyay. "Loss of Mitochondrial Localization of Human FANCG Causes Defective FANCJ Helicase." Molecular and Cellular Biology 40, no. 23 (September 28, 2020). http://dx.doi.org/10.1128/mcb.00306-20.
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ABSTRACT Fanconi anemia (FA) is a unique DNA damage repair pathway. To date, 22 genes have been identified that are associated with the FA pathway. A defect in any of those genes causes genomic instability, and the patients bearing the mutation become susceptible to cancer. In our earlier work, we identified that Fanconi anemia protein G (FANCG) protects the mitochondria from oxidative stress. In this report, we have identified eight patients having a mutation (C.65G>C), which converts arginine at position 22 to proline (p.Arg22Pro) in the N terminus of FANCG. The mutant protein, hFANCGR22P, is able to repair the DNA and able to retain the monoubiquitination of FANCD2 in the FANCGR22P/FGR22P cell. However, it lost mitochondrial localization and failed to protect mitochondria from oxidative stress. Mitochondrial instability in the FANCGR22P cell causes the transcriptional downregulation of mitochondrial iron-sulfur cluster biogenesis protein frataxin (FXN) and the resulting iron deficiency of FA protein FANCJ, an iron-sulfur-containing helicase involved in DNA repair.
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Cheng, Kaiying, and Dale B. Wigley. "DNA translocation mechanism of an XPD family helicase." eLife 7 (December 6, 2018). http://dx.doi.org/10.7554/elife.42400.
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The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily two helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5’−3’ direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bound to single-stranded DNA (ssDNA) in the presence and absence of an ATP analogue (ADP•BeF3), that suggest a mechanism for 5’−3’ translocation along the ssDNA substrate. This proposed mechanism has implications for how those enzymes of the XPD family that recognise bulky DNA lesions might stall at these as the first step in initiating DNA repair. Biochemical data reveal roles for conserved residues that are mutated in human diseases.
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Lowran, Kaitlin, Colin Wu, and Ingrid Petersen. "Abstract P487: Impact Of Oxidative Dna Damage On Heart Health." Circulation Research 129, Suppl_1 (September 3, 2021). http://dx.doi.org/10.1161/res.129.suppl_1.p487.
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The accumulation of DNA damage in human cardiomyocytes causes apoptosis which can lead to heart failure or other cardiovascular diseases. Although the effects of oxidative stress on heart health and on the DNA Damage Response network are well-known, the two fields have evolved as separate areas of research. The precise impact of oxidative DNA damage on cardiomyocyte contractile function still remains poorly understood. The human FANCJ helicase participates in multiple DNA repair pathways, including interstrand crosslink repair and double-stranded break repair. We have shown previously that FANCJ targets and unfolds 8-oxoguanine modified DNA secondary structures that arise from oxidative damage. We predict that human cardiomyocytes expressing mutations of FANCJ would be more susceptible to oxidative DNA damage and will negatively influence their contractile motion. To test this, hiPSC-CMs were treated with hydrogen peroxide, camptothecin, or bleomycin to induce different forms of DNA damage. The relative abundance of single-stranded DNA breaks and double-stranded DNA breaks were determined by modified comet assays, while contractile function was monitored using video-based detection methods. Cells that overexpress FANCJ protein were able to overcome the chemical stress from hydrogen peroxide. On the contrary, cells that produce a FANCJ K141/K142AA variant, which was previously characterized in the lab, resulted in a hypersensitivity to double-stranded DNA breaks. Based on this evidence, FANCJ plays a vital role in alleviating the effects of oxidative stress. Our long-term goal is to use the established methods to develop functional assays that characterize the cardiovascular risks of other FANCJ variants. These assays can be used to develop screening methods to identify patients who may be predisposed to FANCJ-associated cardiovascular diseases.
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Lowran, Kaitlin A., Mikayla McCarthy, Laura Campbell, and Colin Wu. "Abstract 500: Impact of Oxidative Stress and Fancj on Heart Health." Circulation Research 127, Suppl_1 (July 31, 2020). http://dx.doi.org/10.1161/res.127.suppl_1.500.
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G-quadruplexes (or G4s) are structures formed by guanine-rich nucleic acid sequences. G4s must be promptly unfolded in cells; otherwise, they can interfere with DNA replication, RNA transcription, and other essential processes. Guanines bases are susceptible to oxidative stress forming 8-oxoguanines (8oxoG). Although 8oxoG-modified DNA sequences can still fold into stable G4s, it is not known how 8oxoG4s are repaired in human cells. In our previous study, we have shown that the FANCJ DNA helicase targets G4s using an AKKQ amino acid motif. Here, we have examined the interactions of FANCJ with various 8oxoG4s using biolayer interferometry and fluorescence spectroscopy. We show that a FANCJ AKKQ peptide alone can recognize G4s with an affinity of 3.7uM. Moreover, this motif binds to 8oxoG4s with greater affinities of 1.3 to 2.3uM. A detailed description of the mechanisms by which 8oxoG4s are repaired is essential for understanding how human hearts respond to oxidative stress. To test the importance of FANCJ AKKQ-G4 interactions in cells, we measured the total extent of oxidative DNA damage in human cardiac cells by single-cell electrophoresis. Cells that overexpress FANCJ can readily overcome the chemical stress induced by hydrogen peroxide treatment and the G4-stabilizing compound telomestatin. On the contrary, cells that produce a FANCJ AAAQ mutant, which cannot interact with G4s, resulted in an accumulation of 8oxoG4s. Based on this evidence, FANCJ plays an important role to alleviate the damage caused by oxidative stress. In future experiments, we plan to further examine the cardiovascular risks of DNA damage caused by FANCJ malfunctions.
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Brosh, Robert M., and Yuliang Wu. "An emerging picture of FANCJ’s role in G4 resolution to facilitate DNA replication." NAR Cancer 3, no. 3 (July 2, 2021). http://dx.doi.org/10.1093/narcan/zcab034.
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Abstract A well-accepted hallmark of cancer is genomic instability, which drives tumorigenesis. Therefore, understanding the molecular and cellular defects that destabilize chromosomal integrity is paramount to cancer diagnosis, treatment and cure. DNA repair and the replication stress response are overarching paradigms for maintenance of genomic stability, but the devil is in the details. ATP-dependent helicases serve to unwind DNA so it is replicated, transcribed, recombined and repaired efficiently through coordination with other nucleic acid binding and metabolizing proteins. Alternatively folded DNA structures deviating from the conventional anti-parallel double helix pose serious challenges to normal genomic transactions. Accumulating evidence suggests that G-quadruplex (G4) DNA is problematic for replication. Although there are multiple human DNA helicases that can resolve G4 in vitro, it is debated which helicases are truly important to resolve such structures in vivo. Recent advances have begun to elucidate the principal helicase actors, particularly in cellular DNA replication. FANCJ, a DNA helicase implicated in cancer and the chromosomal instability disorder Fanconi Anemia, takes center stage in G4 resolution to allow smooth DNA replication. We will discuss FANCJ’s role with its protein partner RPA to remove G4 obstacles during DNA synthesis, highlighting very recent advances and implications for cancer therapy.
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Lee, Wei Ting C., Yandong Yin, Michael J. Morten, Peter Tonzi, Pam Pam Gwo, Diana C. Odermatt, Mauro Modesti, et al. "Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling." Nature Communications 12, no. 1 (May 5, 2021). http://dx.doi.org/10.1038/s41467-021-22830-9.
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AbstractGuanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.
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Sathish, Sneha, Leslie Morris, and Linda Bloom. "Expression and Characterization of YoaA, a Putative Helicase in Bacteria, Involved in Repairing Blocks to DNA Replication." UF Journal of Undergraduate Research 20, no. 3 (May 2, 2019). http://dx.doi.org/10.32473/ufjur.v20i3.106284.
Full textAbstract:
While cells have efficient pathways for repairing damage to DNA, some DNA damage avoids repair and is found in DNA replication. Because the DNA polymerases that replicate the genome are high fidelity enzymes, DNA damage blocks DNA synthesis and ultimately progression of the replication fork. It is known that cells would not be able to survive without mechanisms to repair these replication blocks and to restart replication. With a genetic screen, our collaborators at Lovett laboratory, using 3’azidothymidine (AZT) as a tool to inhibit replication, identified a novel gene in Escherichia coli, yoaA, which was required along with holC to provide cells with tolerance to AZT. HolC is a protein subunit of the E.coli DNA polymerase III holoenzyme that does the bulk of synthesis during DNA replication, and yoaA binds HolC. Based on sequence, the yoaA gene encodes an iron-sulfur (Fe-S) helicases. E.coli contains a second Fe-S helicase, DinG, and human cells contain four Fe-S helicases, XPD, FANCJ, RTEL1, and ChlR1, that are involved in DNA repair. The overall goal of this project is to express YoaA protein in soluble form and characterize its biochemical activities to determine how YoaA aids in DNA replication fork repair. In our initial studies, we made fluorescent labeled DNA for helicase assays and tested them which allowed us to work towards our long term goals of determining: 1) whether YoaA is DNA helicase and what are the best substrates for YoaA, and 2) how HolC affects YoaA activities. We have subcloned the yoaA gene into different expression vectors to express YoaA with and without affinity tags, and to co-express YoaA with HolC. We are developing strategies to purify YoaA alone and in a complex with HolC. We have subcloned the yoaA gene into different expression vectors to express yoaA with pCOLADuet. We are developing strategies to purify YoaA alone. The initial results working towards this are presented.