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Nucleus
Break induced replication is a specific type of DNA repair that has a co-opted role in telomere e... more Break induced replication is a specific type of DNA repair that has a co-opted role in telomere extension by telomerase-negative cancer cells. This Alternative Lengthening of Telomeres (or "ALT") is required for viability in approximately 10% of all carcinomas, but up to 50% of soft-tissue derived sarcomas. In several recent studies, we and others demonstrate that expression and activity of FANCM, a DNA translocase protein, is essential for the viability of ALT-associated cancers. Here we provide a summary of how and why FANCM depletion leads to deletion of ALT-controlled cancers, predominantly through a hyperactivation of break-induced replication. We also discuss how FANCM can and has been targeted in cancer cell killing, including potential opportunities in ALT and other genetic backgrounds. Mechanism of Alternative Lengthening of Telomeres (ALT) Telomeres consist of 5'-TTAGGG-3' repeats at the ends of chromosomes. They are shortened by 50-200 base pairs per cell cycle 1 because the DNA replication machinery cannot catalyze the complete replication of linear molecules (the "end replication problem" 2). Telomere shortening creates a barrier to tumorigenesis and limits the lifespan of transformed cells 3. The majority of carcinomas and almost all hematologic malignancies subvert this barrier through activation of a reverse transcriptase enzyme called telomerase that overcomes telomere shortening by synthesizing new telomeric DNA. Telomerase is a target in cancer therapy 4, 5 , however not all cancers use telomerase to maintain telomeres. Instead, at least 40% of sarcomas use a recombination/replication-based mechanism, called Alternative-lengthening of telomeres (ALT) 6. Sarcomas that use ALT are more refractory to treatment and carry a higher risk of
Co-transcriptional R-loops arise from physiological or aberrant stalling of RNA polymerase, leadi... more Co-transcriptional R-loops arise from physiological or aberrant stalling of RNA polymerase, leading to formation of stable DNA:RNA hybrids. Unresolved R-loops can promote genome instability. Here, we show that the Fanconi anemia- and breast cancer-associated FANCM protein can directly unwind DNA-RNA hybrids from co-transcriptional R-loops in vitro. FANCM processively unwinds both short and long R-loops, irrespective of sequence, topology or coating by replication protein A. R-loops can also be unwound in the same assay by the yeast and bacterial orthologs of FANCM, Mph1 and RecG, indicating an evolutionary conserved function. Consistent with this biochemical activity of FANCM, we show that FANCM deficient cells are sensitive to drugs that stabilize R-loop formation. Our work reveals a mechanistic basis for R-loop metabolism that is critical for genome stability.
Scientific reports, Jan 9, 2017
Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organism... more Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundat...
Nucleus
Break induced replication is a specific type of DNA repair that has a co-opted role in telomere e... more Break induced replication is a specific type of DNA repair that has a co-opted role in telomere extension by telomerase-negative cancer cells. This Alternative Lengthening of Telomeres (or "ALT") is required for viability in approximately 10% of all carcinomas, but up to 50% of soft-tissue derived sarcomas. In several recent studies, we and others demonstrate that expression and activity of FANCM, a DNA translocase protein, is essential for the viability of ALT-associated cancers. Here we provide a summary of how and why FANCM depletion leads to deletion of ALT-controlled cancers, predominantly through a hyperactivation of break-induced replication. We also discuss how FANCM can and has been targeted in cancer cell killing, including potential opportunities in ALT and other genetic backgrounds. Mechanism of Alternative Lengthening of Telomeres (ALT) Telomeres consist of 5'-TTAGGG-3' repeats at the ends of chromosomes. They are shortened by 50-200 base pairs per cell cycle 1 because the DNA replication machinery cannot catalyze the complete replication of linear molecules (the "end replication problem" 2). Telomere shortening creates a barrier to tumorigenesis and limits the lifespan of transformed cells 3. The majority of carcinomas and almost all hematologic malignancies subvert this barrier through activation of a reverse transcriptase enzyme called telomerase that overcomes telomere shortening by synthesizing new telomeric DNA. Telomerase is a target in cancer therapy 4, 5 , however not all cancers use telomerase to maintain telomeres. Instead, at least 40% of sarcomas use a recombination/replication-based mechanism, called Alternative-lengthening of telomeres (ALT) 6. Sarcomas that use ALT are more refractory to treatment and carry a higher risk of
Co-transcriptional R-loops arise from physiological or aberrant stalling of RNA polymerase, leadi... more Co-transcriptional R-loops arise from physiological or aberrant stalling of RNA polymerase, leading to formation of stable DNA:RNA hybrids. Unresolved R-loops can promote genome instability. Here, we show that the Fanconi anemia- and breast cancer-associated FANCM protein can directly unwind DNA-RNA hybrids from co-transcriptional R-loops in vitro. FANCM processively unwinds both short and long R-loops, irrespective of sequence, topology or coating by replication protein A. R-loops can also be unwound in the same assay by the yeast and bacterial orthologs of FANCM, Mph1 and RecG, indicating an evolutionary conserved function. Consistent with this biochemical activity of FANCM, we show that FANCM deficient cells are sensitive to drugs that stabilize R-loop formation. Our work reveals a mechanistic basis for R-loop metabolism that is critical for genome stability.
Scientific reports, Jan 9, 2017
Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organism... more Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundat...