Formation Of Noncanonical Structures Research Articles

Chasing Particularities of Guanine- and Cytosine-Rich DNA Strands.

By substitution of natural nucleotides by their abasic analogs (i.e., 1′,2′-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first intron of N-myc gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson–Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.

Guanine-guided time-resolved luminescence recognition of DNA modification and i-motif formation by a terbium(III)-platinum(II) complex

Site-specific recognition of DNA modification or the formation of noncanonical structures has important applications in molecular biology, disease diagnosis, and gene expression analysis. In this study, we introduce a guanine-guided sensing tool using a terbium(III)-platinum(II) complex (TPC) as a time-resolved luminescence probe to site-specifically recognize DNA modification and i-motif formation in aqueous solution. The probe is composed of a TbIII center as the luminescent reporter and two PtII units as the receptor for guanine (G) nucleobase. TPC exhibits remarkable reaction selectivity for guanine nucleotides over other nucleotides, giving rise to a significant increase in luminescence. The luminescence enhancement of TPC is mainly attributed to an energy transfer from G base to the TbIII center after the specific coordination of PtII with N7 of guanine (N7-G), which would be facilitated by the phosphates through promoting the departure of coordinated water and bringing G closer to TbIIIvia noncovalent interactions. Based on such sensing feature, the enhanced luminescence of TPC sensitized by G nucleotides can correspondingly decrease upon N7-G modifications of DNA or i-motif formation through constructing simple guanine-guided sensing tools. This probe would provide a useful strategy for site-specific recognition of DNA for extensive purposes.

Identification of G-quadruplex forming sequences in three manatee papillomaviruses.

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Drastic stability change of X-X mismatch in d(CXG) trinucleotide repeat disorders under molecular crowding condition

The trinucleotide repeat d(CXG) (X = A, C, G or T) is the most common sequence causing repeat expansion disorders. The formation of non-canonical structures, such as hairpin structures with X-X mismatches, has been proposed to affect gene expression and regulation, which are important in pathological studies of these devastating neurological diseases. However, little information is available regarding the thermodynamics of the repeat sequence under crowded cellular conditions where many non-canonical structures such as G-quadruplexes are highly stabilized, while duplexes are destabilised. In this study, we investigated the different stabilities of X-X mismatches in the context of internal d(CXG) self-complementary sequences in an environment with a high concentration of cosolutes to mimic the crowding conditions in cells. The stabilities of full-matched duplexes and duplexes with A-A, G-G, and T-T mismatched base pairs under molecular crowding conditions were notably decreased compared to under dilute conditions. However, the stability of the DNA duplex with a C-C mismatch base pair was only slightly destabilised. Investigating different stabilities of X-X mismatches in d(CXG) sequences is important for improving our understanding of the formation and transition of multiple non-canonical structures in trinucleotide repeat diseases, and may provide insights for pathological studies and drug development.

Unique Length-Dependent Biophysical Properties of Repetitive DNA.

Expansion of a trinucleotide repeat (TNR) sequence is the molecular signature of several neurological disorders. The formation of noncanonical structures by the TNR sequence is proposed to contribute to the expansion mechanism. Furthermore, it is known that the propensity for expansion increases with repeat length. In this work, we use calorimetry to describe the thermodynamic parameters (ΔH, TΔS, and ΔG) of the noncanonical stem-loop hairpins formed by the TNR sequences (CAG)n and (CTG)n, as well as the canonical (CAG)n/(CTG)n duplexes, for n = 6-14. Using a thermodynamic cycle, we calculated the same thermodynamic parameters describing the process of converting from noncanonical stem-loop hairpins to a canonical duplex. In addition to these thermodynamic analyses, we used spectroscopic techniques to determine the rate at which the noncanonical structures convert to duplex and the activation enthalpy ΔH(⧧) describing this process. We report that the thermodynamic parameters of unfolding the stem-loop (CTG)n and (CAG)n hairpins, along with the thermodynamic and kinetic properties of hairpin to duplex conversion, do not proportionally correspond to the increase in length, but rather show a unique pattern that depends on whether the sequence has an even or odd number of repeats.

Conformational polymorphysm of G-rich fragments of DNA ALU-repeats. I. Potential noncanonical structures

In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

Trinucleotide Repeat DNA Alters Structure To Minimize the Thermodynamic Impact of 8-Oxo-7,8-dihydroguanine

In the phenomenon of trinucleotide repeat (TNR) expansion, an important interplay exists between DNA damage repair of 8-oxo-7,8-dihydroguanine (8-oxoG) and noncanonical structure formation. We show that TNR DNA adapts its structure to accommodate 8-oxoG. Using chemical probe analysis, we find that CAG repeats composing the stem-loop arm of a three-way junction alter the population of structures in response to 8-oxoG by positioning the lesion at or near the loop. Furthermore, we find that oligonucleotides composed of odd-numbered repeat sequences, which form populations of two structures, will also alter their structure to place 8-oxoG in the loop. However, sequences with an even number of repeats do not display this behavior. Analysis by differential scanning calorimetry indicates that when the lesion is located within the loop, there are no significant changes to the thermodynamic parameters as compared to the DNA lacking 8-oxoG. This contrasts with the enthalpic destabilization observed when 8-oxoG is base-paired to C and indicates that positioning 8-oxoG in the loop avoids the thermodynamic penalty associated with 8-oxoG base-pairing. Since formation of stem-loop hairpins is proposed to facilitate TNR expansion, these results highlight the importance of defining the structural consequences of DNA damage.

The role of ATP in the function of human ORC4 protein

Human ORC4 protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of ATPases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.

The role of ATP in the function of human ORC4 protein

Human ORC4 protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of ATPases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.