scholarly journals RAD51AP1 mediates RAD51 activity through nucleosome interaction

2020 ◽  
Author(s):  
Elena Pires ◽  
Neelam Sharma ◽  
Platon Selemenakis ◽  
Bo Wu ◽  
Yuxin Huang ◽  
...  
Keyword(s):  
Genome Instability ◽  
Loop Formation ◽  
Synaptic Complex ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Nucleosome Core Particles ◽  
Dna Capture ◽  
Dna Template ◽  
Template Dna

AbstractRAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination DNA repair pathway (HR). Loss of RAD51AP1 leads to defective HR, genome instability and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of the donor DNA, synaptic complex assembly and displacement-loop formation when tested with synthetic, nucleosome-free DNA substratesin vitro. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. How RAD51AP1 functions as an HR activator in the context of chromatin has remained unclear.In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs). We identified a C-terminal region in RAD51AP1 and its previously mapped DNA binding domain as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. We show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1directlyassists the RAD51-mediated search of donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

scholarly journals suGF1 binds in the major groove of its oligo(dG).oligo(dC) recognition sequence and is excluded by a positioned nucleosome core.

1994 ◽  
Vol 14 (2) ◽  
pp. 1410-1418 ◽  
Author(s):  
D Patterton ◽  
J Hapgood
Keyword(s):  
Sea Urchin ◽  
Intergenic Region ◽  
Divalent Cations ◽  
Major Groove ◽  
Recognition Sequence ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Developmental Gene Regulation ◽  
Dna Complex

We have elsewhere reported the purification of a poly(dG).poly(dC)-binding nuclear protein (suGF1) from sea urchin embryos (J. Hapgood and D. Patterton, Mol. Cell. Biol. 14:this issue, 1994). We proposed that suGF1 may be a member of a family of G-string factors involved in developmental gene regulation, possibly via alterations in chromatin structure. In this article, we characterize the binding of purified suGF1 to 11 contiguous Gs in the H1-H4 intergenic region of a sea urchin early histone gene battery in vitro. It is shown that suGF1-DNA binding is dependent on ionic strength and requires divalent cations. Purified suGF1 forms discrete protein-DNA multimers, consistent with suGF1-suGF1 interactions. In a model for the suGF1-DNA complex derived from our footprinting and methylation interference data, suGF1 contacts the Gs in the major groove as well as one of the bordering phosphate backbones. The data are consistent with the direction of curvature of the DNA in the suGF1-DNA complex being the same as that preferred by the free DNA and exhibited by the DNA when bent around a positioned nucleosome core in vitro. However, on the basis of steric considerations, the binding of suGF1 and that of the histone octamer are predicted to be mutually exclusive. We show that suGF1 is indeed unable to bind to the G string when occupied by a histone octamer located in the major in vitro positioning frame in the H1-H4 intergenic region.

scholarly journals Mechanism of inhibition of Escherichia coli RNA polymerase by captan

1982 ◽  
Vol 201 (1) ◽  
pp. 145-151 ◽  
Author(s):  
J W Dillwith ◽  
R A Lewis
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Mechanism Of Inhibition ◽  
Dna Binding Site ◽  
Rna Polymerase Activity ◽  
Dna Template ◽  
Template Dna

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.

Different mechanisms for in vitro formation of nucleosome core particles

Biochemistry ◽  
1991 ◽  
Vol 30 (20) ◽  
pp. 5022-5032 ◽  
Author(s):  
Anna M. Aragay ◽  
Xavier Fernandez-Busquets ◽  
Joan Ramon Daban
Keyword(s):  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Core Particles

scholarly journals The prenucleosome, a stable conformational isomer of the nucleosome

2015 ◽  
Vol 29 (24) ◽  
pp. 2563-2575 ◽  
Author(s):  
Jia Fei ◽  
Sharon E. Torigoe ◽  
Christopher R. Brown ◽  
Mai T. Khuong ◽  
George A. Kassavetis ◽  
...  
Keyword(s):  
Motor Protein ◽  
Upstream Region ◽  
Entry And Exit ◽  
Core Particle ◽  
Nucleosome Core Particle ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Conformational Isomer

Chromatin comprises nucleosomes as well as nonnucleosomal histone–DNA particles. Prenucleosomes are rapidly formed histone–DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone–DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone–DNA species in the cell.

suGF1 binds in the major groove of its oligo(dG).oligo(dC) recognition sequence and is excluded by a positioned nucleosome core

1994 ◽  
Vol 14 (2) ◽  
pp. 1410-1418
Author(s):  
D Patterton ◽  
J Hapgood
Keyword(s):  
Sea Urchin ◽  
Intergenic Region ◽  
Divalent Cations ◽  
Major Groove ◽  
Recognition Sequence ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Developmental Gene Regulation ◽  
Dna Complex

We have elsewhere reported the purification of a poly(dG).poly(dC)-binding nuclear protein (suGF1) from sea urchin embryos (J. Hapgood and D. Patterton, Mol. Cell. Biol. 14:this issue, 1994). We proposed that suGF1 may be a member of a family of G-string factors involved in developmental gene regulation, possibly via alterations in chromatin structure. In this article, we characterize the binding of purified suGF1 to 11 contiguous Gs in the H1-H4 intergenic region of a sea urchin early histone gene battery in vitro. It is shown that suGF1-DNA binding is dependent on ionic strength and requires divalent cations. Purified suGF1 forms discrete protein-DNA multimers, consistent with suGF1-suGF1 interactions. In a model for the suGF1-DNA complex derived from our footprinting and methylation interference data, suGF1 contacts the Gs in the major groove as well as one of the bordering phosphate backbones. The data are consistent with the direction of curvature of the DNA in the suGF1-DNA complex being the same as that preferred by the free DNA and exhibited by the DNA when bent around a positioned nucleosome core in vitro. However, on the basis of steric considerations, the binding of suGF1 and that of the histone octamer are predicted to be mutually exclusive. We show that suGF1 is indeed unable to bind to the G string when occupied by a histone octamer located in the major in vitro positioning frame in the H1-H4 intergenic region.

scholarly journals Emerging roles for R-loop structures in the management of topological stress

2020 ◽  
Vol 295 (14) ◽  
pp. 4684-4695 ◽  
Author(s):  
Frederic Chedin ◽  
Craig J. Benham
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Genome Instability ◽  
Dna Topology ◽  
Loop Structure ◽  
Loop Modeling ◽  
Loop Formation ◽  
Dna Structures ◽  
Recent Developments ◽  
Dna Template

R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria, and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops' potential biological roles under both normal and aberrant conditions.

scholarly journals Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site

2009 ◽  
Vol 30 (1) ◽  
pp. 146-159 ◽  
Author(s):  
Deepankar Roy ◽  
Zheng Zhang ◽  
Zhengfei Lu ◽  
Chih-Lin Hsieh ◽  
Michael R. Lieber
Keyword(s):  
Somatic Hypermutation ◽  
Initiation Site ◽  
Loop Formation ◽  
Class Switch ◽  
Dna Strands ◽  
Dna Strand ◽  
Template Dna ◽  
Rna Transcript

ABSTRACT Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template DNA strand, resulting in an R loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance between the promoter and the R-loop-supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA transcript is downstream from the 5′ end of the transcript, the ability of this portion of the transcript to anneal to the template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases because the G-rich portion of the RNA is now closer to the 5′ end of the transcript. Short G-clustered regions can act as RIZs and reduce the distance-induced suppression of R-loop formation. Supercoiled DNA is known to favor transient separation of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick can serve as a strong RIZ, even in regions with no G richness. This has important implications for class switch recombination and somatic hypermutation and possibly for other biological processes in transcribed regions.

scholarly journals FANCM-family branchpoint translocases remove co-transcriptional R-loops

2018 ◽  
Author(s):  
Charlotte Hodson ◽  
Julienne J O’Rourke ◽  
Sylvie van Twest ◽  
Vincent J Murphy ◽  
Elyse Dunn ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Rna Polymerase ◽  
Fanconi Anemia ◽  
Genome Stability ◽  
Genome Instability ◽  
Protein A ◽  
Loop Formation ◽  
Biochemical Activity ◽  
Mechanistic Basis

AbstractCo-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.

scholarly journals Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA

2021 ◽  
Author(s):  
Katie H. Jameson ◽  
Christian J. Rudolph ◽  
Michelle Hawkins
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Processing Enzymes ◽  
Replication Fork Progression ◽  
Evolutionary Advantage ◽  
Domains Of Life ◽  
Dna Template ◽  
Template Dna

ABSTRACTThe complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the ‘replication fork trap’ region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-ter and termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

scholarly journals The Silent Information Regulator 3 Protein, SIR3p, Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt

2008 ◽  
Vol 28 (11) ◽  
pp. 3563-3572 ◽  
Author(s):  
Steven J. McBryant ◽  
Christine Krause ◽  
Christopher L. Woodcock ◽  
Jeffrey C. Hansen
Keyword(s):  
Chromatin Fiber ◽  
Chromatin Binding ◽  
Nucleosome Core ◽  
Naked Dna ◽  
Sir Proteins ◽  
Nucleosome Core Particles ◽  
Chromatin Fibers ◽  
Binding Determinants ◽  
Insight Into

ABSTRACT The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or defined nucleosomal arrays, and the resulting complexes were analyzed by electrophoretic shift assays, sedimentation velocity, and electron microscopy. SIR3p bound avidly to all three types of templates. SIR3p loading onto its nucleosomal sites in chromatin produced thickened condensed fibers that retained a beaded morphology. At higher SIR3p concentrations, individual nucleosomal arrays formed oligomeric suprastructures bridged by SIR3p oligomers. When condensed SIR3p-bound chromatin fibers were incubated in Mg2+, they folded and oligomerized even further to produce hypercondensed higher-order chromatin structures. Collectively, these results define how SIR3p may function as a chromatin architectural protein and provide new insight into the interplay between endogenous and protein-mediated chromatin fiber condensation pathways.

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