The Experts below are selected from a list of 15951 Experts worldwide ranked by ideXlab platform
Gijs J. L. Wuite - One of the best experts on this subject based on the ideXlab platform.
-
DNA Translocations in Real-Time: Insights in Non-Homologous End Joining
Biophysical Journal, 2016Co-Authors: Ineke Brouwer, Gerrit Sitters, Andrea Candelli, Mauro Modesti, Erwin J.g. Peterman, Gijs J. L. WuiteAbstract:DNA translocations are key factors in deregulating cell growth. Currently known translocations are estimated to drive at least 20% of cancer cases. In vitro single-molecule assays of DNA translocations are technically challenging since translocations occurs often at random sites of distinct DNA elements. Here we present a dual-DNA manipulation and fluorescence visualization assay using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where we reconstitute an in vitro model of a translocation between two gene-sized DNA molecules by first inducing DNA break at controlled locations and subsequently observing, in real time, the repair of DNA catalyzed by two core elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation of a bridge between broken DNA molecule having extraordinary strength and stability.
-
DNA Translocations in Real-Time: Insights into Non-Homologous End Joining Pathway
Biophysical Journal, 2015Co-Authors: Andrea Candelli, Ineke Brouwer, Gerrit Sitters, Mauro Modesti, Erwin J.g. Peterman, Stephanie J. Heerema, Gijs J. L. WuiteAbstract:DNA translocations are key factors in deregulating cell growth. Currently known translocations are estimated to drive at least 20% of cancer cases. In vitro single-molecule assays of DNA translocations are technically challenging since translocations occurs often at random sites of distinct DNA elements. Here we present a dual-DNA manipulation and fluorescence visualization assay using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where we reconstitute an in vitro model of a translocation between two gene-sized DNA molecules by first inducing DNA break at controlled locations and subsequently observing, in real time, the repair of DNA catalyzed by two core elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation of a bridge between broken DNA molecule having extraordinary strength and stability.
Joseph J. Loparo - One of the best experts on this subject based on the ideXlab platform.
-
XLF acts as a flexible connector during Non-Homologous End Joining.
eLife, 2020Co-Authors: Sean M. Carney, Andrew T. Moreno, Sadie C Piatt, Metztli Cisneros-aguirre, Felicia Wednesday Lopezcolorado, Jeremy M. Stark, Joseph J. LoparoAbstract:Non-Homologous End Joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA Ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in End synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for End Joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe End synapsis in real-time show that this defect is due to a failure to closely align DNA Ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.
-
XLF acts as a flexible connector during Non-Homologous End Joining
2020Co-Authors: Sean M. Carney, Andrew T. Moreno, Sadie C Piatt, Metztli Cisneros-aguirre, Felicia Wednesday Lopezcolorado, Jeremy M. Stark, Joseph J. LoparoAbstract:Abstract Non-Homologous End Joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA Ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in End synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for End Joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe End synapsis in real-time show that this defect is due to a failure to closely align DNA Ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.
-
A Mechanism to Minimize Errors during Non-Homologous End Joining.
Molecular cell, 2019Co-Authors: Benjamin M. Stinson, Johannes C. Walter, Andrew T. Moreno, Joseph J. LoparoAbstract:Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by Non-Homologous End Joining (NHEJ), which directly ligates DNA Ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA Ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that End processing requires the formation of a "short-range synaptic complex" in which DNA Ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of End processing to a ligation-competent complex ensures that DNA Ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.
-
A mechanism to minimize errors during Non-Homologous End Joining
2019Co-Authors: Benjamin M. Stinson, Johannes C. Walter, Andrew T. Moreno, Joseph J. LoparoAbstract:SUMMARY Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by Non-Homologous End-Joining (NHEJ), which directly ligates DNA Ends. NHEJ has the potential to be highly mutagenic because it employs DNA polymerases, nucleases, and other enzymes that modify incompatible DNA Ends to allow their ligation. Using a biochemical system that recapitulates key features of cellular NHEJ, we show that End-processing requires formation of a “short-range synaptic complex” in which DNA Ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of End processing to a ligation-competent complex ensures that DNA Ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.
-
Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts.
Methods in enzymology, 2017Co-Authors: Thomas G.w. Graham, Johannes C. Walter, Joseph J. LoparoAbstract:Non-Homologous End Joining (NHEJ) repairs the majority of DNA double-strand breaks in human cells, yet the detailed order of events in this process has remained obscure. Here, we describe how to employ Xenopus laevis egg extract for the study of NHEJ. The egg extract is easy to prepare in large quantities, and it performs efficient End Joining that requires the core End Joining proteins Ku, DNA-PKcs, XLF, XRCC4, and DNA ligase IV. These factors, along with the rest of the soluble proteome, are present at Endogenous concentrations, allowing mechanistic analysis in a system that begins to approximate the complexity of cellular End Joining. We describe an ensemble assay that monitors covalent Joining of DNA Ends and fluorescence assays that detect Joining of single pairs of DNA Ends. The latter assay discerns at least two discrete intermediates in the bridging of DNA Ends.
David J. Chen - One of the best experts on this subject based on the ideXlab platform.
-
DNA double strand break repair via Non-Homologous End-Joining
Translational cancer research, 2013Co-Authors: Anthony J. Davis, David J. ChenAbstract:DNA double-stranded breaks (DSB) are among the most dangerous forms of DNA damage. Unrepaired DSBs results in cells undergoing apoptosis or senescence whereas mis-processing of DSBs can lead to genomic instability and carcinogenesis. One important pathway in eukaryotic cells responsible for the repair of DSBs is Non-Homologous End-Joining (NHEJ). In this review we will discuss the interesting new insights into the mechanism of the NHEJ pathway and the proteins which mediate this repair process. Furthermore, the general role of NHEJ in promoting genomic stability will be discussed.
-
Role of SUMO:SIM-mediated protein–protein interaction in Non-Homologous End Joining
Oncogene, 2010Co-Authors: Jeremy M. Stark, David J. Chen, David K. Ann, Y. ChenAbstract:Although post-translational modifications by the small ubiquitin-like modifiers (SUMO) are known to be important in DNA damage response, it is unclear whether they have a role in double-strand break (DSB) repair by Non-Homologous End Joining (NHEJ). Here, we analyzed various DSB repair pathways upon inhibition of SUMO-mediated protein-protein interactions using peptides that contain the SUMO-interaction motif (SIM) and discriminate between mono- and SUMO-chain modifications. The SIM peptides specifically inhibit NHEJ as shown by in vivo repair assays and radio-sensitivity of cell lines deficient in different DSB repair pathways. Furthermore, mono-SUMO, instead of SUMO-chain, modifications appear to be involved in NHEJ. Immunoprecipitation experiments also showed that the SIM peptide interacted with SUMOylated Ku70 after radiation. This study is the first to show an important role for SUMO:SIM-mediated protein-protein interactions in NHEJ, and provides a mechanistic basis for the role of SIM peptide in sensitizing genotoxic stress of cancer cells.
-
The Endless tale of Non-Homologous End-Joining.
Cell research, 2008Co-Authors: Eric Weterings, David J. ChenAbstract:DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and Non-Homologous End-Joining (NHEJ). The NHEJ process utilizes enzymes that capture both Ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.
-
Role of Non-Homologous End Joining (NHEJ) in maintaining genomic integrity
DNA Repair, 2006Co-Authors: Sandeep Burma, Benjamin P C Chen, David J. ChenAbstract:Of the various types of DNA damage that can occur within the mammalian cell, the DNA double strand break (DSB) is perhaps the most dangerous. DSBs are typically induced by intrinsic sources such as the by products of cellular metabolism or by extrinsic sources such as X-rays or gamma-rays and chemotherapeutic drugs. It is becoming increasing clear that an inability to respond properly to DSBs will lead to genomic instability and promote carcinogenesis. The mammalian cell, therefore, has in place several mechanisms that can respond rapidly to DSBs. In this review, we focus on the role of one such mechanism, the Non-Homologous End Joining (NHEJ) pathway of DSB repair, in maintaining genome integrity and preventing carcinogenesis.
Ineke Brouwer - One of the best experts on this subject based on the ideXlab platform.
-
DNA Translocations in Real-Time: Insights in Non-Homologous End Joining
Biophysical Journal, 2016Co-Authors: Ineke Brouwer, Gerrit Sitters, Andrea Candelli, Mauro Modesti, Erwin J.g. Peterman, Gijs J. L. WuiteAbstract:DNA translocations are key factors in deregulating cell growth. Currently known translocations are estimated to drive at least 20% of cancer cases. In vitro single-molecule assays of DNA translocations are technically challenging since translocations occurs often at random sites of distinct DNA elements. Here we present a dual-DNA manipulation and fluorescence visualization assay using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where we reconstitute an in vitro model of a translocation between two gene-sized DNA molecules by first inducing DNA break at controlled locations and subsequently observing, in real time, the repair of DNA catalyzed by two core elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation of a bridge between broken DNA molecule having extraordinary strength and stability.
-
DNA Translocations in Real-Time: Insights into Non-Homologous End Joining Pathway
Biophysical Journal, 2015Co-Authors: Andrea Candelli, Ineke Brouwer, Gerrit Sitters, Mauro Modesti, Erwin J.g. Peterman, Stephanie J. Heerema, Gijs J. L. WuiteAbstract:DNA translocations are key factors in deregulating cell growth. Currently known translocations are estimated to drive at least 20% of cancer cases. In vitro single-molecule assays of DNA translocations are technically challenging since translocations occurs often at random sites of distinct DNA elements. Here we present a dual-DNA manipulation and fluorescence visualization assay using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where we reconstitute an in vitro model of a translocation between two gene-sized DNA molecules by first inducing DNA break at controlled locations and subsequently observing, in real time, the repair of DNA catalyzed by two core elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation of a bridge between broken DNA molecule having extraordinary strength and stability.
Andrea Candelli - One of the best experts on this subject based on the ideXlab platform.
-
DNA Translocations in Real-Time: Insights in Non-Homologous End Joining
Biophysical Journal, 2016Co-Authors: Ineke Brouwer, Gerrit Sitters, Andrea Candelli, Mauro Modesti, Erwin J.g. Peterman, Gijs J. L. WuiteAbstract:DNA translocations are key factors in deregulating cell growth. Currently known translocations are estimated to drive at least 20% of cancer cases. In vitro single-molecule assays of DNA translocations are technically challenging since translocations occurs often at random sites of distinct DNA elements. Here we present a dual-DNA manipulation and fluorescence visualization assay using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where we reconstitute an in vitro model of a translocation between two gene-sized DNA molecules by first inducing DNA break at controlled locations and subsequently observing, in real time, the repair of DNA catalyzed by two core elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation of a bridge between broken DNA molecule having extraordinary strength and stability.
-
DNA Translocations in Real-Time: Insights into Non-Homologous End Joining Pathway
Biophysical Journal, 2015Co-Authors: Andrea Candelli, Ineke Brouwer, Gerrit Sitters, Mauro Modesti, Erwin J.g. Peterman, Stephanie J. Heerema, Gijs J. L. WuiteAbstract:DNA translocations are key factors in deregulating cell growth. Currently known translocations are estimated to drive at least 20% of cancer cases. In vitro single-molecule assays of DNA translocations are technically challenging since translocations occurs often at random sites of distinct DNA elements. Here we present a dual-DNA manipulation and fluorescence visualization assay using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where we reconstitute an in vitro model of a translocation between two gene-sized DNA molecules by first inducing DNA break at controlled locations and subsequently observing, in real time, the repair of DNA catalyzed by two core elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation of a bridge between broken DNA molecule having extraordinary strength and stability.
