Triplex H-DNA structure: the long and winding road from the discovery to its role in human disease

Abstract

H-DNA is an intramolecular DNA triplex formed by homopurine/homopyrimidine mirror repeats. Since its discovery, the field has advanced from characterizing the structure in vitro to discovering its existence and role in vivo. H-DNA interacts with cellular machinery in unique ways, stalling DNA and RNA polymerases and causing genome instability. The foundational S1 nuclease and chemical probing technologies originally used to show H-DNA formation have been updated and combined with genome-wide sequencing methods for large-scale mapping of secondary structures. There is evidence for triplex H-DNA's role in polycystic kidney disease (PKD), cancer, and numerous repeat expansion diseases (REDs). In PKD, an H-DNA forming repeat region within the PKD1 gene stalls DNA replication and induces fragility. H-DNA-forming repeats in various genes have a role in cancer; the most well-studied examples involve H-DNA-mediated fragility causing translocations in multiple lymphomas. Lastly, H-DNA-forming repeats have been implicated in four REDs: Friedreich's ataxia, GAA-FGF14-related ataxia, X-linked Dystonia Parkinsonism, and cerebellar ataxia, neuropathy and vestibular areflexia syndrome. In this review, we summarize H-DNA's discovery and characterization, evidence for its existence and function in vivo, and the field's current knowledge on its role in physiology and pathology.

Graphical Abstract

Figure 1.

Isoforms of triplex DNA. Schematic of H-r DNA, H-y DNA, H-yr DNA/Nodule DNA, and sticky DNA. Black lines indicate non-repetitive DNA. Red and blue lines indicate the homopurine and homopyrimidine strands of a mirror repeat, respectively. 5′ and 3′ are not annotated because the structures can be formed with either orientation of 5′ and 3′. (A) H-r DNA: One half of the homopurine strand of a hPu/hPy mirror repeat folds back to be antiparallel to its other half and binds via reverse Hoogsteen hydrogen bonding in the major groove of the duplex, leaving half of the homopyrimidine strand single-stranded. (B) H-y DNA: One half of the homopyrimidine strand of a hPu/hPy mirror repeat folds back to be antiparallel to its other half and binds via Hoogsteen hydrogen bonding to the purine strand in the major groove of the duplex, leaving half of the homopurine strand single-stranded. (C) H-yr DNA/Nodule DNA: A combination of H-r DNA and H-y DNA, leaving very little single-strandedness. (D) Sticky DNA: Half of this H-r triplex is made up by one half of a hPu/hPy mirror repeat, while the other half is distant from the first, separated by a stretch of double-stranded DNA, but is oriented antiparallel to the first sequence. Created in BioRender. Hisey, J. (2024) https://biorender.com/p95w386.

Figure 2.

Timeline of H-DNA discovery. Schematic outlining the major discoveries that led to a full understanding of triplex H-DNA’s structure. Synthetic three-stranded ribonucleotide complex (4); Hoogsteen and reverse Hoogsteen hydrogen bonding (6,7); Synthetic dsDNA:ssRNA and triple-stranded complexes (8–11); Supercoiling- and low pH-dependent S1 hypersensitivity found in hPu/hPy sequences; structural theories arose (10,21–33); 2D gels show structural transition correlates to unwound state (33,41,42); Mirror repeat nature proven (43); H-r DNA triplex described (34); Chemical probing supports H-y DNA triplex structure (44–48); why H-y3 versus H-y5 isoform formed (49,50); AFM of H-DNA (71). Created in BioRender. Hisey, J. (2024) https://biorender.com/m31s510.

Figure 3.

Base triads that stabilize triplex formation. (A) TA*T and CG*C⁺ base triads with Watson-Crick and Hoogsteen (*) hydrogen bonding. (B) TA*A and CG*G base triads with Watson-Crick and reverse Hoogsteen (*) hydrogen bonding. Created in BioRender. Hisey, J. (2024) https://biorender.com/f14l364.

Figure 4.

Two-dimensional gel electrophoresis of topoisomers and its use in triplex H-DNA discovery. (A) Schematic of a 2D gel separating various topoisomers of a given plasmid. Blue circles represent negatively supercoiled plasmids, red circles represent positively supercoiled plasmids, and gray circles represent plasmids without supercoiling. Numbers indicate the number of supercoils the plasmid has and if they are positive or negative supercoils. In the first dimension, plasmids with the same absolute value of their number of supercoils run through a gel identically: positively and negatively supercoiled DNA topoisomers move more quickly through the gel with an increasing number of supercoils. In the second dimension, the gel is run in the presence of chloroquine, which unwinds DNA, thereby causing negatively supercoiled plasmids to become less supercoiled and therefore migrate slower and positively supercoiled plasmids to become more supercoiled and therefore migrate faster, thereby separating the negatively supercoiled plasmids (blue) from their positively supercoiled counterparts (red). (B) Schematic of a 2D gel of a plasmid containing (GA)₁₆ from a sea urchin histone gene spacer region where a structural transition (black bracket) equivalent to a complete unwinding of (GA)₁₆ was detected; figure adapted from results found in Figure 3 of (41). Created in BioRender. Hisey, J. (2024) https://BioRender.com/z79v169.

Figure 5.

Models of H-r triplex formation during cellular processes, leading to polymerase stalling, and other downstream consequences. (A) Polymerase stalling due to triplex formed during polymerization on a single-stranded template. Black lines indicate non-repetitive DNA. Red and blue lines indicate the homopurine and homopyrimidine strands of a mirror repeat, respectively. (B) Polymerase stalling due to triplex formed during strand displacement. (C) Preformed triplex in supercoiled DNA causing replication fork stalling. (D) Triplex formed during replication leading to replication fork stalling. (E) Replication fork stalling leading to fork reversal. (F) H-loop is a composite structure arising during transcription, in which the RNA transcript binds to the single-stranded portion of H-DNA formed upstream of the elongating RNAP. The green line indicates the mRNA transcript. The blue oval-shaped structure is RNAP. Created in BioRender. Hisey, J. (2024) https://biorender.com/o41t359.

Figure 6.

Models of DNA repair machinery cleaving H-DNA. Triplex H-DNA structure with scissors indicating where the labeled nucleases are proposed to cut. Black lines indicate non-repetitive DNA. Red and blue lines indicate the homopurine and homopyrimidine strands of a mirror repeat, respectively. Figure based off of the findings referenced in the text (85,95). Created in BioRender. Hisey, J. (2024) https://BioRender.com/i80j609.

Figure 7.

Transient cellular processes promoting triplex formation. (A) RNAP induces positive supercoiling ahead and negative supercoiling behind as it progresses from left to right in the diagram. Negative supercoiling behind RNAP promotes triplex formation. Black lines indicate non-repetitive DNA. Red and blue lines indicate the homopurine and homopyrimidine strands of a mirror repeat, respectively. The blue oval-shaped structure is RNAP. (B) Negative supercoiling forms upon nucleosome (blue cylinder) removal, which then promotes triplex formation. Processes that unwind the duplex or otherwise lead to ssDNA such as (C) replication, (D) transcription (green line represents mRNA transcript) or (E) DNA repair (DSB with a hPu-rich 3′ overhang or gap fill-in) can promote triplex formation. Created in BioRender. Hisey, J. (2024) https://BioRender.com/o82v488.

Figure 8.

A model of FRDA’s triplex H-DNA-based pathogenic mechanism. During cellular processes that unwind duplex DNA, (GAA)_exp repeats in the first intron of the Frataxin (FXN) gene may form a triplex H-DNA secondary structure. This may happen during transcription and concurrent R-loop formation (also called an H-loop) may help to stabilize the H-DNA structure and stall transcription at the repeats. Proteins such as those able to bind the repeats and chromatin modifiers (dark blue and green structures) are then recruited to the repeats, leading to heterochromatinization of the repeats that spreads upstream, leading to FXN promoter silencing. Transcription start stie is represented by the angled arrow. RNAP is represented by the blue oval-shaped structure. Histones are represented by aqua cylindrical structures. Created in BioRender. Hisey, J. (2024) https://biorender.com/a21m828.