A key interaction with RPA orients XPA in NER complexes

Abstract The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD–RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

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Critical region and metal–nonmetal transition in expanded fluid mercury: advanced evaluation of small-angle X-ray scattering data

Journal of Applied Crystallography ◽

10.1107/s0021889809055113 ◽

2010 ◽

Vol 43 (2) ◽

pp. 244-249 ◽

Cited By ~ 9

Author(s):

Wilhelm Ruland ◽

Friedrich Hensel

Keyword(s):

Small Angle ◽

Critical Region ◽

Structural Model ◽

Volume Fraction ◽

Critical Density ◽

Scattering Data ◽

Two Phase ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

The small-angle X-ray scattering data of expanded fluid mercury published in the literature were evaluated using a modified form of the Teubner–Strey equation for microemulsions together with the general treatment of two-phase systems according to Porod. The parameters obtained in the critical region and the metal–nonmetal (M–NM) transition are evidence of a nanoemulsion composed of M and NM domains. The structure of this emulsion is characterized by density, volume fraction and size parameters (average chord length, polydispersity) of the domains. On the basis of these parameters, a structural model for fluid mercury in the liquid–vapour critical region and the M–NM transition is developed. Analysis of the relationship between the volume fraction of the M domains and the electrical conductivity reveals that a percolation transition occurs, with a threshold located near the liquid–vapour critical density. This observation is consistent with recent theoretical developments.

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A New Epistasis Group for the Repair of DNA Damage in Bacteriophage T4: Replication Repair

Genetics ◽

10.1093/genetics/115.3.405 ◽

1987 ◽

Vol 115 (3) ◽

pp. 405-417

Author(s):

Joseph T Wachsman ◽

John W Drake

Keyword(s):

Dna Replication ◽

Excision Repair ◽

Bacteriophage T4 ◽

Repair Process ◽

Dna Helicase ◽

Host Cells ◽

Repair System ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

Gene 32

ABSTRACT The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication.

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Insight into dynamics of APOBEC3G protein in complexes with DNA assessed by high speed AFM

10.1101/581793 ◽

2019 ◽

Author(s):

Yangang Pan ◽

Luda S. Shlyakhtenko ◽

Yuri L. Lyubchenko

Keyword(s):

High Speed ◽

Molecular Mechanisms ◽

Catalytic Domain ◽

Structural Information ◽

Dynamic Structure ◽

Time Lapse ◽

Ssdna Binding ◽

Binding Domains ◽

Ssdna Binding Protein ◽

Dumbbell Structure

AbstractAPOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome. A3G has two zing-binding domains: the N-terminal domain (NTD), which efficiently binds ssDNA, and the C-terminal catalytic domain (CTD), which supports deaminase activity of A3G. Until now, structural information on A3G has lacked, preventing elucidation of the molecular mechanisms underlying its interaction with ssDNA and deaminase activity. We have recently built computational model for the full-length A3G monomer and validated its structure by data obtained from time-lapse High-Speed Atomic Force Microscopy (HS AFM). Here time-lapse HS AFM was applied to directly visualize the structure and dynamics of A3G in complexes with ssDNA. Our results demonstrate a highly dynamic structure of A3G, where two domains of the protein fluctuate between compact globular and extended dumbbell structures. Quantitative analysis of our data revealed a substantial increase in the number of A3G dumbbell structures in the presence of the DNA substrate, suggesting the interaction of A3G with the ssDNA substrate stabilizes this dumbbell structure. Based on these data, we developed a model explaining the interaction of globular and dumbbell structures of A3G with ssDNA and suggested a possible role of the dumbbell structure in A3G function.

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Phosphoproteomics Reveals a Distinct Mode of Mec1/ATR Signaling in Response to DNA End Hyper-Resection

10.1101/2020.04.17.028118 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ethan J. Sanford ◽

Vitor M. Faça ◽

Stephanie C. Vega ◽

William J. Comstock ◽

Marcus B. Smolka

Keyword(s):

Dna Repair ◽

Scaffolding Protein ◽

Genome Maintenance ◽

Distinct Mode ◽

Checkpoint Signaling ◽

Ssdna Binding ◽

Binding Domains ◽

Ssdna Binding Protein ◽

Atr Kinase ◽

Dna Ends

ABSTRACTThe Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, although how it promotes context-dependent signaling and DNA repair remains elusive. Here we uncovered a specialized mode of Mec1/ATR signaling triggered by the extensive nucleolytic processing (resection) of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct mode of Mec1 signaling triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

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Structure of the amorphous titania precursor phase of N-doped photocatalysts

RSC Advances ◽

10.1039/d0ra08886b ◽

2021 ◽

Vol 11 (15) ◽

pp. 8619-8627

Author(s):

I. E. Grey ◽

P. Bordet ◽

N. C. Wilson

Keyword(s):

Thermal Analyses ◽

Real Space ◽

Ammonia Solution ◽

Scattering Data ◽

X Ray ◽

X Ray Scattering ◽

Amorphous Titania ◽

Precursor Phase ◽

Titania Precursor ◽

Titanyl Sulphate

Amorphous titania samples prepared by ammonia solution neutralization of titanyl sulphate have been characterized by chemical and thermal analyses, and with reciprocal-space and real-space fitting of wide-angle synchrotron X-ray scattering data.

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In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽

10.1093/genetics/159.4.1617 ◽

2001 ◽

Vol 159 (4) ◽

pp. 1617-1630

Author(s):

Suk-Won Jin ◽

Nancy Arno ◽

Adam Cohen ◽

Amy Shah ◽

Qijin Xu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Germ Cell ◽

Rna Binding ◽

Dominant Negative ◽

Missense Mutations ◽

Biochemical Tests ◽

Cell Fates ◽

Cytoplasmic Polyadenylation ◽

Binding Domains ◽

Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

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Coupling In-Situ Techniques to Analyze Zinc Deposition and Dissolution for Energy Storage Applications

MRS Proceedings ◽

10.1557/opl.2012.1734 ◽

2013 ◽

Vol 1491 ◽

Cited By ~ 2

Author(s):

Jayme Keist ◽

Christine Orme ◽

Frances Ross ◽

Dan Steingart ◽

Paul Wright ◽

...

Keyword(s):

Liquid Electrolyte ◽

Scattering Data ◽

Zinc Deposition ◽

Ionic Liquid Electrolyte ◽

Force Microscopy ◽

In Situ Techniques ◽

X Ray Scattering ◽

Atomic Force ◽

Afm Analysis

ABSTRACTThis investigation describes preliminary results of in-situ analysis of zinc deposition within an ionic liquid electrolyte utilizing electrochemical atomic force microscopy (EC AFM). From the AFM analysis, the morphology of the zinc deposition was analyzed by quantifying the surface roughness using height-height correlation functions. These results will be used to analyze the scattering data obtained from zinc deposition analysis utilizing an electrochemical ultra-small angle x-ray scattering (EC USAXS). The goal of this research is to link the early nucleation and growth behavior to the formation of detrimental morphologies.

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The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

Nature Communications ◽

10.1038/s41467-020-20818-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ann-Marie K. Shorrocks ◽

Samuel E. Jones ◽

Kaima Tsukada ◽

Carl A. Morrow ◽

Zoulikha Belblidia ◽

...

Keyword(s):

Replication Stress ◽

Bloom Syndrome ◽

Holliday Junctions ◽

Replication Forks ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

Dna Replication Stress ◽

Dna End Resection ◽

Stalled Replication Forks

AbstractThe Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

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Global Small-Angle X-ray Scattering Data Analysis of Triacylglycerols in the Molten State (Part I)

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.8b06704 ◽

2018 ◽

Vol 122 (45) ◽

pp. 10320-10329 ◽

Cited By ~ 7

Author(s):

Amin Sadeghpour ◽

Marjorie Ladd Parada ◽

Josélio Vieira ◽

Megan Povey ◽

Michael Rappolt

Keyword(s):

Data Analysis ◽

Small Angle ◽

Scattering Data ◽

Molten State ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

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Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

Journal of Nucleic Acids ◽

10.4061/2011/529828 ◽

2011 ◽

Vol 2011 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Aishwarya Prakash ◽

Fabien Kieken ◽

Luis A. Marky ◽

Gloria E. O. Borgstahl

Keyword(s):

Dna Replication ◽

Protein A ◽

Thermal Stabilization ◽

Replication Protein A ◽

Replication Protein ◽

Unique Problem ◽

Ssdna Binding ◽

Binding Domains ◽

G Quadruplex ◽

Stabilization Effect

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′-TAGGGGAAGGGTTGGAGTGGGTT-3′called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+Gq23 has a less stable (TMlowered by ∼20∘C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+form of Gq23 very efficiently, but complete unfolding is not observed with the K+form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA.

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