Rdh54/Tid1 inhibits Rad51-Rad54-mediated D-loop formation and limits D-loop length

Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

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Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

10.1101/2020.05.20.106641 ◽

2020 ◽

Cited By ~ 2

Author(s):

Shanaya Shital Shah ◽

Stella Hartono ◽

Aurèle Piazza ◽

Vanessa Som ◽

William Wright ◽

...

Keyword(s):

Binding Sites ◽

Loop Length ◽

Loop Formation ◽

Independent Manner ◽

Potential Binding ◽

D Loop ◽

Strand Invasion ◽

Type Switch ◽

Dna Strand

ABSTRACTDisplacement loops (D-loops) are intermediates formed during homologous recombination that play a pivotal role in the fidelity of repair. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54 is also present in somatic cells where its function is less understood. While Rdh54 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54-mediated invasions. Here, we show that Rdh54 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54 uniquely restricts the lengths of Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast, whereas Rdh54 expression is suppressed in diploids. We propose that Rdh54 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54 acts as a physical roadblock to Rad54’s translocation activity, limiting D-loop formation and D-loop length.

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Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2162 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2162-2171 ◽

Cited By ~ 66

Author(s):

C S Madsen ◽

S C Ghivizzani ◽

W W Hauswirth

Keyword(s):

Mitochondrial Dna ◽

Protein Binding ◽

Loop Formation ◽

Sequence Element ◽

Conserved Sequence ◽

Loop Region ◽

In Organello ◽

Close Proximity ◽

D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

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Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

10.1101/2020.05.20.106617 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shanaya Shital Shah ◽

Stella Hartono ◽

Frédéric Chédin ◽

Wolf-Dietrich Heyer

Keyword(s):

Real Time ◽

Single Molecule ◽

Detection Methods ◽

Loop Formation ◽

Bisulfite Treatment ◽

Loop Detection ◽

D Loop ◽

Donor Choice ◽

The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

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Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

10.1101/2021.06.22.449520 ◽

2021 ◽

Author(s):

Terren Chang ◽

Xi Long ◽

Shankar Shastry ◽

Joseph William Parks ◽

Michael D Stone

Keyword(s):

Single Molecule ◽

Mechanical Analysis ◽

Magnetic Tweezers ◽

Repeat Sequence ◽

Single Stranded Dna ◽

Telomere Repeat ◽

D Loop ◽

Human Telomere ◽

Strand Invasion

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

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INSILICO STUDIES OF OXIME PRODRUG OF GLICLAZIDE AGAINST SULPHONYLUREA RECEPTORS (SUR 1)

International Journal of Current Pharmaceutical Research ◽

10.22159/ijcpr.2017v9i4.20968 ◽

2017 ◽

Vol 9 (4) ◽

pp. 100

Author(s):

Surendran Vijayaraj ◽

Kannekanti Chaithanya Veena

Keyword(s):

Binding Sites ◽

Pancreatic Beta Cells ◽

Molecular Targets ◽

Docking Studies ◽

Docking Analysis ◽

Potential Binding ◽

In Silico Study ◽

Molecular Docking Analysis ◽

Autodock 4.2

Objective: Objective of the study is to perform a molecular docking analysis of novel oxime prodrug of gliclazide against SUR1 receptor.Methods: Sulfonylurea receptors (SUR) are membrane proteins which are the molecular targets of the sulfonylurea class of anti-diabetic drugs whose mechanism of action is to promote insulin release from pancreatic beta cells. Oxime prodrug of gliclazide a better soluble derivative of gliclazide is used for enhancement of bioavailability of gliclazide. Autodock 4.2 software was used for docking studies. Ligand 2D structures were drawn using ChemDraw Ultra 7.0. Binding sites, docking poses and interactions of the ligand with SUR1 receptors were studied by pymol software.Results: The docking studies suggest that potential binding sites of oxime prodrug of gliclazide exhibiting all the major interactions such as hydrogen bonding, hydrophobic interaction and electrostatic interaction with GLU43, LEU11, LEU 40, ILE17 GLU 68, GLN72 residues of SUR1. The binding energy of complexes are also found to be minimal forming stable complexes.Conclusion: In silico study of oxime prodrug of gliclazide conforms, the binding of oxime prodrug of glicalzide with SUR1 receptors which effectively controls the release insulin to regulate plasma glucose concentrations. Hence, the oxime prodrug of gliclazide could be a potent anti-diabetic target molecule which may be worth for further in vitro and in vivostudies.

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AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

Journal of Bacteriology ◽

10.1128/jb.00314-15 ◽

2015 ◽

Vol 197 (24) ◽

pp. 3788-3796 ◽

Cited By ~ 3

Author(s):

Takayuki Kuge ◽

Haruhiko Teramoto ◽

Masayuki Inui

Keyword(s):

Corynebacterium Glutamicum ◽

Binding Sites ◽

Large Scale ◽

Transcriptional Regulator ◽

Scale Production ◽

Primary Role ◽

Promoter Regions ◽

Independent Manner ◽

Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

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Decision letter: Rdh54/Tid1 inhibits Rad51-Rad54-mediated D-loop formation and limits D-loop length

10.7554/elife.59112.sa1 ◽

2020 ◽

Author(s):

Anna Malkova ◽

Andrew J Deans

Keyword(s):

Loop Length ◽

Loop Formation ◽

D Loop

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TraY and Integration Host Factor oriT Binding Sites and F Conjugal Transfer: Sequence Variations, but Not Altered Spacing, Are Tolerated

Journal of Bacteriology ◽

10.1128/jb.01783-06 ◽

2007 ◽

Vol 189 (10) ◽

pp. 3813-3823 ◽

Cited By ~ 15

Author(s):

Sarah L. Williams ◽

Joel F. Schildbach

Keyword(s):

Conformational Changes ◽

Binding Sites ◽

Integration Host Factor ◽

Host Factor ◽

Conjugative Plasmid ◽

Sequence Specificity ◽

Single Stranded Region ◽

Plasmid Mobilization ◽

Dna Strand

ABSTRACT Bacterial conjugation is the process by which a single strand of a conjugative plasmid is transferred from donor to recipient. For F plasmid, TraI, a relaxase or nickase, binds a single plasmid DNA strand at its specific origin of transfer (oriT) binding site, sbi, and cleaves at a site called nic. In vitro studies suggest TraI is recruited to sbi by its accessory proteins, TraY and integration host factor (IHF). TraY and IHF bind conserved oriT sites sbyA and ihfA, respectively, and bend DNA. The resulting conformational changes may propagate to nic, generating the single-stranded region that TraI can bind. Previous deletion studies performed by others showed transfer efficiency of a plasmid containing F oriT decreased progressively as increasingly longer segments, ultimately containing both sbyA and ihfA, were deleted. Here we describe our efforts to more precisely define the role of sbyA and ihfA by examining the effects of multiple base substitutions at sbyA and ihfA on binding and plasmid mobilization. While we observed significant decreases in in vitro DNA-binding affinities, we saw little effect on plasmid mobilization even when sbyA and ihfA variants were combined. In contrast, when half or full helical turns were inserted between the relaxosome protein-binding sites, mobilization was dramatically reduced, in some cases below the detectable limit of the assay. These results are consistent with TraY and IHF recognizing sbyA and ihfA with limited sequence specificity and with relaxosome proteins requiring proper spacing and orientation with respect to each other.

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Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2162-2171.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2162-2171

Author(s):

C S Madsen ◽

S C Ghivizzani ◽

W W Hauswirth

Keyword(s):

Mitochondrial Dna ◽

Protein Binding ◽

Loop Formation ◽

Sequence Element ◽

Conserved Sequence ◽

Loop Region ◽

In Organello ◽

Close Proximity ◽

D Loop

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Cooperative Pho2-Pho4 Interactions at thePHO5 Promoter Are Critical for Binding of Pho4 to UASp1 and for Efficient Transactivation by Pho4 at UASp2

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2629 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2629-2639 ◽

Cited By ~ 53

Author(s):

Slobodan Barbaric ◽

Martin Münsterkötter ◽

Colin Goding ◽

Wolfram Hörz

Keyword(s):

Binding Sites ◽

Promoter Activity ◽

Regulatory Elements ◽

Striking Difference ◽

Homeodomain Protein ◽

Interaction Domain ◽

Independent Manner ◽

Activity Measurements

ABSTRACT The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes toPHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

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