scholarly journals DNA triple-helix formation on nucleosome-bound poly(dA)·poly(dT) tracts

1998 ◽  
Vol 333 (2) ◽  
pp. 259-267 ◽  
Author(s):  
Philip M. BROWN ◽  
Keith R. FOX
Keyword(s):  
Hydroxyl Radical ◽  
Dna Fragments ◽  
Protein Surface ◽  
Surface Hydroxyl ◽  
Helix Formation ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Radical Cleavage ◽  
The Stability ◽  
Hydroxyl Radical Footprinting

We have used DNase I and hydroxyl-radical footprinting to examine the formation of intermolecular DNA triple helices on nucleosome-bound DNA fragments containing An·Tn tracts. We found that it is possible to form triplexes on these nucleosome-bound DNAs, but the stability of the complexes depends on the orientation of the A tract with respect to the protein surface. Hydroxyl-radical cleavage of these complexes suggests that the DNA fragments are still associated with the nucleosome. However, the phased cleavage pattern is lost in the vicinity of the triplex, suggesting that the DNA has locally moved away from the protein surface.

scholarly journals Stability of an RNA•DNA–DNA triple helix depends on base triplet composition and length of the RNA third strand

2019 ◽  
Vol 47 (14) ◽  
pp. 7213-7222 ◽  
Author(s):  
Charlotte N Kunkler ◽  
Jacob P Hulewicz ◽  
Sarah C Hickman ◽  
Matthew C Wang ◽  
Phillip J McCown ◽  
...  
Keyword(s):  
Relative Stability ◽  
Triple Helix ◽  
Multiple Factors ◽  
Dna And Rna ◽  
Helix Formation ◽  
Canonical Base ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Dna Triple Helix ◽  
The Stability

AbstractRecent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA–DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA–DNA triple helices is not well understood beyond the canonical U•A–T and C•G–C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X–Y (where Z = C, U, A, G and X–Y = A–T, G–C, T–A, C–G), in an RNA•DNA–DNA triple helix was determined. The canonical U•A–T and C•G–C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA–DNA triple helix can tolerate up to two consecutive non-canonical A•G–C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA–DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA–DNA and RNA•RNA–RNA triple helices was distinctly different from those in RNA•DNA–DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.

scholarly journals Nucleosome core particles inhibit DNA triple helix formation

1996 ◽  
Vol 319 (2) ◽  
pp. 607-611 ◽  
Author(s):  
Philip M BROWN ◽  
Keith R FOX
Keyword(s):  
Target Site ◽  
Nucleosome Core ◽  
Dna Triplexes ◽  
Helix Formation ◽  
Nucleosome Core Particles ◽  
Dnase I Footprinting ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Dna Fragment ◽  
Core Particles

We have used DNase I footprinting to examine the formation of DNA triple helices at target sites on DNA fragments that have been reconstituted with nucleosome core particles. We show that a 12 bp homopurine target site, located 45 bp from the end of the 160 bp tyrT(46A) fragment, cannot be targeted with either parallel (CT-containing) or antiparallel (GT-containing) triplex-forming oligonucleotides when reconstituted on to nucleosome core particles. Binding is not facilitated by the presence of a triplex-binding ligand. However, both parallel and antiparallel triplexes could be formed on a truncated DNA fragment in which the target site was located closer to the end of the DNA fragment. We suggest that intermolecular DNA triplexes can only be formed on those DNA regions that are less tightly associated with the protein core.

The detrimental effect of orotic acid substitution in the peptide nucleic acid strand on the stability of PNA2:NA triple helices

2005 ◽  
Vol 83 (10) ◽  
pp. 1731-1740 ◽  
Author(s):  
Robert HE Hudson ◽  
Filip Wojciechowski
Keyword(s):  
Nucleic Acid ◽  
Carboxylic Acid ◽  
Peptide Nucleic Acid ◽  
Triple Helix ◽  
Orotic Acid ◽  
Uv Spectroscopy ◽  
Triplex Formation ◽  
Helix Formation ◽  
Triple Helix Formation ◽  
The Stability

We have investigated the incorporation of C6 derivatives of uracil into polypyrimidine peptide nucleic acid oligomers. Starting with uracil-6-carboxylic acid (orotic acid), a peptide nucleic acid monomer compatible with Fmoc-based synthesis was prepared. This monomer then served as a convertible nucleobase whereupon treatment of the resin-bound methyl orotate containing hexamers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing peptide nucleic acid. Peptide nucleic acid hexamers containing the C6-modified nucleobase hybridized to both poly(riboadenylic acid) and poly(deoxyriboadenylic acid) via triplex formation. Complexes formed with poly(riboadenylic acid) were more stable than those formed with poly(dexoyriboadenylic acid), as measured by temperature-dependent UV spectroscopy. However, both of these complexes were destabilized relative to the complexes formed by an unmodified peptide nucleic acid oligomers. Internal or doubly substituted hexamers are destabilized more strongly than a terminally substituted one, and the type of substitution (carboxamide, ester, carboxylic acid) affects the overall triplex stability. These results clearly show that incorporation of a C6-substituted uracil into polypyrimidine PNA is detrimental to triplex formation. We have also extended this chemistry to incorporate uracil-5-methylcarboxylate into a peptide nucleic acid hexamer. After on-resin conversion of the C5 ester to the 3-(N,N-dimethylamino)propylamide, significant stabilization of the triplex formed with poly(riboadenylic acid) was observed, which illustrates the compatibility of C5 substitution with peptide nucleic acid directed triple helix formation. Key words: peptide nucleic acid, triple helix, orotic acid, orotamide, PNA.

scholarly journals Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides

1994 ◽  
Vol 301 (2) ◽  
pp. 569-575 ◽  
Author(s):  
E Washbrook ◽  
K R Fox
Keyword(s):  
Metal Ion ◽  
Dimethyl Sulphate ◽  
Dnase I ◽  
Target Sequence ◽  
Base Pairs ◽  
Helix Formation ◽  
Dnase I Footprinting ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Dna Triple Helix

We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.

scholarly journals Effect of a triplex-binding ligand on triple helix formation at a site within a natural DNA fragment

1996 ◽  
Vol 314 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Philip M. BROWN ◽  
Amelia DRABBLE ◽  
Keith R. FOX
Keyword(s):  
Target Site ◽  
Target Sequence ◽  
Similar Concentration ◽  
Helix Formation ◽  
Dnase I Footprinting ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Dna Fragment ◽  
A Site ◽  
Formation Of Complexes

We have used DNase I footprinting to examine the effect of a triplex-binding ligand on the formation of parallel intermolecular DNA triple helices at a mixed sequence target site contained within a natural DNA fragment (tyrT). In the presence of 10 μM ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-ylamine), the binding of CTCTTTTTGCTT (12G) to the sequence GAGAAAAATGAA (generating a complex containing 8×T·AT, 1×G·TA and 3×C+·GC triplets) was enhanced 3-fold at pH 5.5. When the oligonucleotide CTCTTTTTTCTT (12T) was substituted for 12G (replacing G·TA with T·TA) there was a large reduction in affinity for the target sequence. However, this was stabilized by about 300-fold in the presence of the ligand, requiring a similar concentration to produce a footprint as 12G in the absence of the ligand. When the sequence of the target site was altered to GAGAAAAAAGAA, generating an uninterrupted run of purines [tyrT(46A)], the binding of 12T (generating a complex containing 9×T·AT, and 3×C+·GC triplets) was enhanced 3-fold by 10 μM of the triplex-binding ligand. However, although the binding of 12G to this sequence, generating a complex containing a G·AT triplet, was much weaker, this too was stabilized by about 30-fold by the ligand, requiring a similar concentration as the perfect matched oligonucleotide (12T) in the absence of the ligand. A secondary, less stable footprint was also observed in these fragments when using either 12T or 12G, which was evident only in the presence of the triplex-binding ligand. This site, which contained a number of triplet mismatches, appears to be related to the formation of four or five central T·AT triplets. This reduction in the stringency of oligonucleotide binding by the triplex-binding ligand promotes the formation of complexes at non-targeted regions but may also have the potential for enabling recognition at sites that contain regions where there are no specific triplet matches.

scholarly journals Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain

2020 ◽  
Vol 295 (29) ◽  
pp. 9959-9973 ◽  
Author(s):  
Ngoc-Duc Doan ◽  
Azade S. Hosseini ◽  
Agata A. Bikovtseva ◽  
Michelle S. Huang ◽  
Andrew S. DiChiara ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Osteogenesis Imperfecta ◽  
Triple Helix ◽  
Helix Formation ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Triple Helical Domain ◽  
Key Aspects ◽  
Intracellular Collagen

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that “C-Pro-only” constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

Triple helix formation by oligopurine-oligopyrimidine DNA fragments

1990 ◽  
Vol 213 (4) ◽  
pp. 833-843 ◽  
Author(s):  
Giorgio Manzini ◽  
Luigi E. Xodo ◽  
Daniela Gasparotto ◽  
Franco Quadrifoglio ◽  
Gijs A. van der Marel ◽  
...  
Keyword(s):  
Triple Helix ◽  
Dna Fragments ◽  
Helix Formation ◽  
Triple Helix Formation
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