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1.
J Biol Chem ; 292(46): 19001-19012, 2017 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-28939774

RESUMEN

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.


Asunto(s)
ADN Bacteriano/metabolismo , AdnB Helicasas/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , ADN Bacteriano/química , AdnB Helicasas/química , Escherichia coli/química , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Alineación de Secuencia , Electricidad Estática
2.
J Biol Chem ; 291(27): 14324-14339, 2016 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-27226550

RESUMEN

Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.


Asunto(s)
ADN Helicasas/metabolismo , ADN/metabolismo , Proteínas Mitocondriales/metabolismo , ADN/química , Daño del ADN , Transferencia Resonante de Energía de Fluorescencia , Humanos , Oxidación-Reducción , Especificidad por Sustrato
3.
Methods ; 108: 79-91, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27068657

RESUMEN

Helicases are proposed to unwind dsDNA primarily by translocating on one strand to sterically exclude and separate the two strands. Hexameric helicases in particular have been shown to encircle one strand while physically excluding the other strand. In this article, we will detail experimental methods used to validate specific interactions with the excluded strand on the exterior surface of hexameric helicases. Both qualitative and quantitative methods are described to identify an excluded strand interaction, determine the exterior interacting residues, and measure the dynamics of binding. The implications of exterior interactions with the nontranslocating strand are discussed and include forward unwinding stabilization, regulation of the unwinding rate, and DNA damage sensing.


Asunto(s)
Daño del ADN/genética , ADN Helicasas/genética , ADN/genética , Ingeniería Genética/métodos , ADN/química , ADN Helicasas/química , Conformación de Ácido Nucleico
4.
Nat Commun ; 15(1): 1250, 2024 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-38341432

RESUMEN

Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/metabolismo , Reparación del ADN , ADN Ligasas/metabolismo , ADN/genética , ADN/metabolismo
5.
Elife ; 92020 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-33289484

RESUMEN

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.


Asunto(s)
Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Cromatografía en Gel , Reparación del ADN por Unión de Extremidades/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Immunoblotting , Óvulo/metabolismo , Alineación de Secuencia , Proteínas de Xenopus/genética , Xenopus laevis/genética
6.
Nat Struct Mol Biol ; 25(9): 877-884, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30177755

RESUMEN

Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.


Asunto(s)
Reparación del ADN por Unión de Extremidades , Animales , Proteínas de Unión al ADN/metabolismo , Dimerización , Humanos , Imagen Óptica , Xenopus laevis
7.
BMC Biophys ; 7: 6, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25132964

RESUMEN

BACKGROUND: A key challenge in interdisciplinary research is choosing the best approach from a large number of techniques derived from different disciplines and their interfaces. RESULTS: To address this challenge in the area of Biophysics and Structural Biology, we have designed a graduate level course to teach students insightful use of experimental biophysical approaches in relationship to addressing biological questions related to biomolecular interactions and dynamics. A weekly seminar and data and literature club are used to compliment the training in class. The course contains wet-laboratory experimental demonstration and real-data analysis as well as lectures, grant proposal preparation and assessment, and student presentation components. Active student participation is mandatory in all aspects of the class. Students prepare materials for the class receiving individual and iterative feedback from course directors and local experts generating high quality classroom presentations. CONCLUSIONS: The ultimate goal of the course is to teach students the skills needed to weigh different experimental approaches against each other in addressing a specific biological question by thinking and executing academic tasks like faculty.

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