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1. Triplex H-DNA Structure: The Long and Winding Road from the Discovery to Its Role in Human Disease

   https://doi.org/10.1093/narmme/ugae024  

   Hisey, Julia A; Masnovo, Chiara; Mirkin, Sergei M (December 2024, NAR Molecular Medicine)

   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. 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, cancer, and numerous repeat expansion diseases. In polycystic kidney disease (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 repeat expansion diseases: Friedreich's ataxia (FRDA), GAA-FGF14-related ataxia, X-linked Dystonia Parkinsonism (XDP), and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS). 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. 

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2. THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions.

   https://doi.org/https://doi.org/10.1101/2022.03.22.4853861  

   Rebecca E. Brown; Xiaofeng A. Su; Stacey Fair; Katherine Wu; Lauren Verra; Robyn Jong; Kristin Andrykovich; Catherine H. Freudenreich (March 2022, bioRxiv)

   Expanded CAG/CTG repeats are sites of DNA damage, leading to changes in repeat length. To determine how ssDNA gap filling affects repeat instability, we inserted (CAG)70 or (CTG)70 repeats into a single-strand annealing (SSA) assay system such that resection and filling in the ssDNA gap would occur across the repeat tract. After resection, when the CTG sequence was the single-stranded template for fill-in synthesis, repeat contractions were elevated and the ssDNA created a fragile site that led to large deletions involving flanking homologous sequences. In contrast, resection was inhibited when CTG was on the resected strand, resulting in repeat expansions. Deleting Rad9, the ortholog of 53BP1, rescued repeat instability and lost viability by increasing resection and fill-in speed. Deletion of Rad51 increased CTG contractions and decreased survival, implicating Rad51 in protecting ssDNA during gap filling. Taken together, DNA sequence within a single-stranded gap determines repair kinetics, fragility, and repeat instability. 

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3. Recurrent DNA nicks drive massive expansions of (GAA) _n repeats

   https://doi.org/10.1073/pnas.2413298121  

   Li, Liangzi; Scott, W_Shem; Khristich, Alexandra_N; Armenia, Jillian_F; Mirkin, Sergei_M (November 2024, Proceedings of the National Academy of Sciences)

   Over 50 hereditary degenerative disorders are caused by expansions of short tandem DNA repeats (STRs). (GAA)_nrepeat expansions are responsible for Friedreich’s ataxia as well as late-onset cerebellar ataxias (LOCAs). Thus, the mechanisms of (GAA)_nrepeat expansions attract broad scientific attention. To investigate the role of DNA nicks in this process, we utilized a CRISPR-Cas9 nickase system to introduce targeted nicks adjacent to the (GAA)_nrepeat tract. We found that DNA nicks 5′ of the (GAA)₁₀₀run led to a dramatic increase in both the rate and scale of its expansion in dividing cells. Strikingly, they also promoted large-scale expansions of carrier- and large normal-size (GAA)_nrepeats, recreating, in a model system, the expansion events that occur in human pedigrees. DNA nicks 3′ of the (GAA)₁₀₀repeat led to a smaller but significant increase in the expansion rate as well. Our genetic analysis implies that in dividing cells, conversion of nicks into double-strand breaks (DSBs) during DNA replication followed by DSB or fork repair leads to repeat expansions. Finally, we showed that 5′ GAA-strand nicks increase expansion frequency in nondividing yeast cells, albeit to a lesser extent than in dividing cells. 

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4. Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae

   https://doi.org/10.1007/978-1-4939-9784-8_5  

   Polleys, Erica J.; Freudenreich, Catherine H (October 2019, Methods in molecular biology)

   Trinucleotide repeats are common in the human genome and can undergo changes in repeat number and cause length-dependent chromosome fragility. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), with variations that allow the role of transcription to be evaluated. The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC). The second fragility assay relies on the fact that a chromosomal break stimulates recombination-mediated repair. A PCR-based assay can be used to evaluate instability of the repeat in the same conditions used to measure repeat fragility. These assays have contributed to understanding the genetic mechanisms that cause chromosome breaks and tract-length changes at unstable trinucleotide repeats. 

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5. Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures

   https://doi.org/10.1093/nar/gkae124  

   Hisey, Julia A.; Radchenko, Elina A.; Mandel, Nicholas H.; McGinty, Ryan J.; Matos-Rodrigues, Gabriel; Rastokina, Anastasia; Masnovo, Chiara; Ceschi, Silvia; Hernandez, Alfredo; Nussenzweig, André; et al (February 2024, Nucleic Acids Research)

   Abstract CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication. 

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