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

H-DNA is an intramolecular DNA triplex formed by homopurine/homopyrimidine mirror repeats. Since its discovery, the field has advanced from characterizing the structure to discovering its existence and role . H-DNA interacts with cellular machinery in unique ways, stalling DNA and RNA polymerases and causing genome instability.

Stabilization of expandable DNA repeats by the replication factor Mcm10 promotes cell viability.

Trinucleotide repeats, including Friedreich's ataxia (GAA) repeats, become pathogenic upon expansions during DNA replication and repair. Here, we show that deficiency of the essential replisome component Mcm10 dramatically elevates (GAA) repeat instability in a budding yeast model by loss of proper CMG helicase interaction. Supporting this conclusion, live-cell microscopy experiments reveal increased replication fork stalling at the repeat in mcm10-1 cells.

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome.

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA.

Massive contractions of myotonic dystrophy type 2-associated CCTG tetranucleotide repeats occur via double-strand break repair with distinct requirements for DNA helicases.

Myotonic dystrophy type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75 to 11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables the selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis.

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

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (AG) repeat in the 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 main pathogenic (AG) and the main nonpathogenic (AG) repeats.

Massive contractions of Myotonic Dystrophy Type 2-associated CCTG tetranucleotide repeats occur via double strand break repair with distinct requirements for helicases.

Myotonic Dystrophy Type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of . The number of CCTG repeats in DM2 patients ranges from 75-11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in that enables selection of large-scale contractions of (CCTG) within the intron of a reporter gene and subsequent genetic analysis.

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S.

S1-END-seq reveals DNA secondary structures in human cells.

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing.

Replication dependent and independent mechanisms of GAA repeat instability.

Trinucleotide repeat instability is a driver of human disease. Large expansions of (GAA) repeats in the first intron of the FXN gene are the cause Friedreich's ataxia (FRDA), a progressive degenerative disorder which cannot yet be prevented or treated. (GAA) repeat instability arises during both replication-dependent processes, such as cell division and intergenerational transmission, as well as in terminally differentiated somatic tissues.

Partners in crime: Tbf1 and Vid22 promote expansions of long human telomeric repeats at an interstitial chromosome position in yeast.

In humans, telomeric repeats (TTAGGG) are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome.

Characteristics and possible mechanisms of formation of microinversions distinguishing human and chimpanzee genomes.

Genomic inversions come in various sizes. While long inversions are relatively easy to identify by aligning high-quality genome sequences, unambiguous identification of microinversions is more problematic. Here, using a set of extra stringent criteria to distinguish microinversions from other mutational events, we describe microinversions that occurred after the divergence of humans and chimpanzees.

Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast.

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich's ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions.

On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability.

Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability.

Large-scale contractions of Friedreich's ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability.

Friedreich's ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA) repeats in the first intron of the gene [V. Campuzano , 271, 1423-1427 (1996)]. In somatic tissues of FRDA patients, (GAA) repeat tracts are highly unstable, with contractions more common than expansions [R.

Experimental System to Study Instability of (CGG) Repeats in Cultured Mammalian Cells.

Expansions of simple trinucleotide repeats, such as (CGG), (CAG) or (GAA), are responsible for more than 40 hereditary disorders in humans including fragile X syndrome, Huntington's disease, myotonic dystrophy, and Friedreich's ataxia. While the mechanisms of repeat expansions were intensively studied for over two decades, the final picture has yet to emerge. It was important, therefore, to develop a mammalian experimental system for studying repeat instability, which would recapitulate repeat instability observed in human pedigrees.

At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences.

Tandem DNA repeats derived from the ancestral (TTAGGG)n run were first detected at chromosome ends of the majority of living organisms, hence the name telomeric DNA repeats. Subsequently, it has become clear that telomeric motifs are also present within chromosomes, and they were suitably called interstitial telomeric sequences (ITSs). It is well known that telomeric DNA repeats play a key role in chromosome stability, preventing end-to-end fusions and precluding the recurrent DNA loss during replication.

Mrc1 and Tof1 prevent fragility and instability at long CAG repeats by their fork stabilizing function.

Fork stabilization at DNA impediments is key to maintaining replication fork integrity and preventing chromosome breaks. Mrc1 and Tof1 are two known stabilizers that travel with the replication fork. In addition to a structural role, Mrc1 has a DNA damage checkpoint function.

Mechanisms of genetic instability caused by (CGG) repeats in an experimental mammalian system.

We developed an experimental system for studying genome instability caused by fragile X (CGG) repeats in mammalian cells. Our method uses a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG) repeats in its 5' UTR, which is integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG) repeats markedly elevated the frequency of reporter inactivation, making cells ganciclovir resistant.