Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability.

Abstract:

:Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. The possibility of manipulating the protein content by using protein depletion procedures makes egg extract a powerful system to study proteins whose inactivation results in cellular lethality. The egg extract has been increasingly used to study DNA damage response and the coordination of DNA replication with DNA repair. The recent development of advanced imaging techniques based on electron microscopy has allowed the characterization of replication intermediates formed in the absence of essential DNA repair proteins. These studies have been important to understand how cells maintain genome stability under unchallenged and stressful conditions. Here, we present a collection of protocols that have been developed to recapitulate DNA damage response activated by chromosome breakage in egg extract and to isolate replication intermediates for electron microscopy analysis using sperm nuclei or more defined genomic substrates.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Sannino V,Pezzimenti F,Bertora S,Costanzo V

doi

10.1016/bs.mie.2017.03.018

subject

Has Abstract

pub_date

2017-01-01 00:00:00

pages

211-232

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(17)30113-1

journal_volume

591

pub_type

杂志文章
  • Peroxiredoxin-6 and NADPH oxidase activity.

    abstract::Peroxiredoxins (Prdxs) are a family of proteins which catalyze the reduction of H2O2 through the interaction of active site cysteine residues. Conserved within all plant and animal kingdoms, the function of these proteins is related to protection from oxidation or participation of signaling through degradation of H2O2...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-405882-8.00008-8

    authors: Ambruso DR

    更新日期:2013-01-01 00:00:00

  • In vivo expression technology for selection of bacterial genes specifically induced in host tissues.

    abstract::We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The syst...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/0076-6879(94)35164-3

    authors: Slauch JM,Mahan MJ,Mekalanos JJ

    更新日期:1994-01-01 00:00:00

  • Glycoprotein maturation and the UPR.

    abstract::Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycos...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385928-0.00010-9

    authors: Hülsmeier AJ,Welti M,Hennet T

    更新日期:2011-01-01 00:00:00

  • Chapter 3. The adenoviral vector angiogenesis/lymphangiogenesis assay.

    abstract::Adenoviral vectors expressing vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A(164)) offer a powerful method for elucidating the mechanisms of pathological angiogenesis and lymphangiogenesis and for evaluating the effectiveness of pro- and anti-angiogenesis therapies. When injected int...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)02803-6

    authors: Nagy JA,Shih SC,Wong WH,Dvorak AM,Dvorak HF

    更新日期:2008-01-01 00:00:00

  • Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling.

    abstract::Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signal...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(04)89005-0

    authors: Hains MD,Siderovski DP,Harden TK

    更新日期:2004-01-01 00:00:00

  • Production and properties of monoclonal antibodies to guinea pig Ia antigens.

    abstract::Although the use of monoclonal antibodies to characterize the Ia antigen system is still in its infancy, the usefulness of this approach is clearly apparent. Since xenogeneic immunization can be used to generate these antibodies, the chances are good that a greater variety of Ia types and determinants will be detected...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(83)92010-4

    authors: Zweig SE,Shevach EM

    更新日期:1983-01-01 00:00:00

  • Flow cytometric analysis of autophagy in living mammalian cells.

    abstract::Autophagy is a major intracellular catabolic pathway induced in response to amino acid starvation. Recent findings implicate it in diverse physiological/pathophysiological events, such as protein and organelle turnover, development, aging, pathogen infection, cell death, and neurodegeneration. However, experimental me...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)03609-4

    authors: Shvets E,Elazar Z

    更新日期:2009-01-01 00:00:00

  • Synthetic biology in Streptomyces bacteria.

    abstract::Actinomycete bacteria of the genus Streptomyces are major producers of bioactive compounds for the biotechnology industry. They are the source of most clinically used antibiotics, as well as of several widely used drugs against common diseases, including cancer . Genome sequencing has revealed that the potential of St...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385075-1.00021-4

    authors: Medema MH,Breitling R,Takano E

    更新日期:2011-01-01 00:00:00

  • Poly(ADP-ribose) polymerase-1 activation during DNA damage and repair.

    abstract::Changes in chromatin structure emanating from DNA breaks are among the most initiating events in the damage response of the cell. In higher eukaryotes, poly(ADP-ribose) polymerase-1 (PARP-1) translates the occurrence of DNA breaks detected by its zinc-finger domain into a signal, poly ADP-ribose, synthesized and ampli...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)09029-4

    authors: Dantzer F,Amé JC,Schreiber V,Nakamura J,Ménissier-de Murcia J,de Murcia G

    更新日期:2006-01-01 00:00:00

  • Human embryo culture.

    abstract::Human embryonic stem cells (hESCs) are derived from preimplantation embryos. Approximately 60% of human embryos are blocked during in vitro development. Although statistics are inconclusive, experience demonstrates that hESCs are more effectively derived from high-quality embryos. In this way, optimal human embryo cul...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)20001-6

    authors: Mercader A,Valbuena D,Simón C

    更新日期:2006-01-01 00:00:00

  • Computational methods in tumor immunology.

    abstract::The remarkable success of cancer immunotherapies, especially the checkpoint blocking antibodies, in a subset of patients has reinvigorated the study of tumor-immune crosstalk and its role in heterogeneity of response. High-throughput sequencing and imaging technologies can help recapitulate various aspects of the tumo...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.01.001

    authors: Bhinder B,Elemento O

    更新日期:2020-01-01 00:00:00

  • Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems.

    abstract::Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been stu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)08012-8

    authors: Reardon JT,Sancar A

    更新日期:2006-01-01 00:00:00

  • Use of sec mutants to define intermediates in protein transport from endoplasmic reticulum.

    abstract::In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion. The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, additi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(92)19028-5

    authors: Rexach MF,Schekman RW

    更新日期:1992-01-01 00:00:00

  • Solving serpin crystal structures.

    abstract::Essentially the same steps are required to solve the crystal structure of a serpin as for any other protein: produce and purify protein, grow crystals, collect diffraction data, find estimates of the phase angles, and then refine and validate the structure. For the phasing step, experimental phasing methods involving ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385950-1.00004-3

    authors: Read RJ,Zhou A,Stein PE

    更新日期:2011-01-01 00:00:00

  • Peripheral clocks and the regulation of cardiovascular and metabolic function.

    abstract::Circadian rhythms generated by cell autonomous biological clocks allow for the appropriate temporal synchronization of physiology and behavior, optimizing the efficiency of biological systems. Circadian oscillators and functions have been uncovered in both central and peripheral tissues. This article describes methodo...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)93027-9

    authors: Rudic RD,Curtis AM,Cheng Y,FitzGerald G

    更新日期:2005-01-01 00:00:00

  • Molecular tools for investigating ANME community structure and function.

    abstract::Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabol...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385112-3.00004-4

    authors: Hallam SJ,Pagé AP,Constan L,Song YC,Norbeck AD,Brewer H,Pasa-Tolic L

    更新日期:2011-01-01 00:00:00

  • Reactions of nitric oxide and oxygen with the regulator of fumarate and nitrate reduction, a global transcriptional regulator, during anaerobic growth of Escherichia coli.

    abstract::The Escherichia coli fumarate and nitrate reductase (FNR) regulator protein is an important transcriptional regulator that controls the expression of a large regulon of more than 100 genes in response to changes in oxygen availability. FNR is active when it acquires a [4Fe-4S](2+) cluster under anaerobic conditions. T...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)37011-0

    authors: Crack JC,Le Brun NE,Thomson AJ,Green J,Jervis AJ

    更新日期:2008-01-01 00:00:00

  • Chapter 11 Supercomplex organization of the yeast respiratory chain complexes and the ADP/ATP carrier proteins.

    abstract::The enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are coassembled into higher ordered supercomplexes within the mitochondrial inner membrane. The cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex is formed by the coassociation of the two electron transport chain complexes, the cytochrome ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)04411-X

    authors: Stuart RA

    更新日期:2009-01-01 00:00:00

  • Studying RNA-RNA and RNA-protein interactions by isothermal titration calorimetry.

    abstract::Isothermal Titration Calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of RNA folding, RNA binding to small molecules, and RNA-protein interactions. The advent of extremely sensitive instrumentation and the increasing availability of ITC in shared facilities have made it in...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)68019-8

    authors: Feig AL

    更新日期:2009-01-01 00:00:00

  • Measurement of mitochondrial turnover and life cycle using MitoTimer.

    abstract::Current methodologies available to quantify changes in mitochondrial turnover are limited to pulse-chase assays or specific assays that quantify mitophagy. Accordingly, new tools that can assess mitochondrial turnover are needed for the study of cellular, subcellular, and spatial parameters of mitochondrial turnover a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-801415-8.00002-3

    authors: Trudeau KM,Gottlieb RA,Shirihai OS

    更新日期:2014-01-01 00:00:00

  • Gene expression omnibus: microarray data storage, submission, retrieval, and analysis.

    abstract::The Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information archives and freely distributes high-throughput molecular abundance data, predominantly gene expression data generated by DNA microarray technology. The database has a flexible design that can handle diverse styles of bot...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)11019-8

    authors: Barrett T,Edgar R

    更新日期:2006-01-01 00:00:00

  • Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance.

    abstract::Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-801122-5.00019-2

    authors: Chang AL,McKeague M,Smolke CD

    更新日期:2014-01-01 00:00:00

  • Purification, analysis, and crystal structure of integrins.

    abstract::Integrins are large modular cell-surface receptors that regulate almost every aspect of cellular function through bidirectional signals transmitted across the lipid bilayer. Regulation of integrin activity is accomplished by complex and still incompletely understood biochemical pathways that modify integrin ligand bin...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)26014-8

    authors: Xiong JP,Goodman SL,Arnaout MA

    更新日期:2007-01-01 00:00:00

  • EPR Studies of Gating Mechanisms in Ion Channels.

    abstract::Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/bs.mie.2014.12.030

    authors: Chakrapani S

    更新日期:2015-01-01 00:00:00

  • Lipidomics: A Corrective Lens for Enzyme Myopia.

    abstract::Classifications and characterizations of specific proteins, such as enzymes, not only allow us to understand biosynthetic and metabolic pathways but they also help to drive our understanding of protein structure and function. How those characterizations are evaluated, however, may change our interpretations and lead u...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2017.06.024

    authors: Bradshaw HB,Leishman E

    更新日期:2017-01-01 00:00:00

  • Peptide sequencing by MALDI 193-nm photodissociation TOF MS.

    abstract::Ultraviolet photodissociation time-of-flight (TOF) mass spectrometry (MS) is described as a method for determination of peptide ion primary structure. Monoisotopic selection and bond-specific activation, combined with the rapidity of TOF MS analysis, render this technique invaluable to the rapidly expanding field of p...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(05)02006-9

    authors: Morgan JW,Hettick JM,Russell DH

    更新日期:2005-01-01 00:00:00

  • Protein labeling for FRET with methoxycoumarin and acridonylalanine.

    abstract::Site-specific protein labeling can be used to monitor protein motions and interactions in real time using Förster resonance energy transfer (FRET). While there are many fluorophores available for protein labeling, few FRET pairs are suitable for monitoring intramolecular protein motions without being disruptive to pro...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.04.008

    authors: Jones CM,Venkatesh Y,Petersson EJ

    更新日期:2020-01-01 00:00:00

  • Imaging single mRNA molecules in yeast.

    abstract::Yeast cells in an isogenic population do not all display the same phenotypes. To study such variation within a population of cells, we need to perform measurements on each individual cell instead of measurements that average out the behavior of a cell over the entire population. Here, we provide the basic concepts and...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(10)70017-3

    authors: Youk H,Raj A,van Oudenaarden A

    更新日期:2010-01-01 00:00:00

  • Fluorescence line narrowing spectroscopy: a tool for studying proteins.

    abstract::Perhaps the most important contribution of FLN is that it provides an experimental approach to relate physical changes in the protein to predicted dynamical behavior. It is clear that the sample is inhomogeneously broadened in a continuous manner, consistent with the damped motion of proteins. At the same time configu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/s0076-6879(97)78007-8

    authors: Vanderkooi JM,Angiolillo PJ,Laberge M

    更新日期:1997-01-01 00:00:00

  • Production of antipolyubiquitin monoclonal antibodies and their use for characterization and isolation of polyubiquitinated proteins.

    abstract::Formation of a Lys48-linked polyubiquitin chain is required for destruction of targeted proteins by the 26S proteasome, whereas formation of a Lys63-linked polyubiquitin chain is required for modulation of protein-protein interaction, enzyme activity, and intracellular localization. In addition, monoubiquitination pla...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)99006-X

    authors: Fujimuro M,Yokosawa H

    更新日期:2005-01-01 00:00:00