APOBEC3A deaminates CTG hairpin loops to promote fragility and instability of expanded CAG/CTG repeats.

CAG/CTG repeats are prone to expansion, causing several inherited human diseases. The initiating sources of DNA damage which lead to inaccurate repair of the repeat tract to cause expansions are not fully understood. Expansion-prone CAG/CTG repeats are actively transcribed and prone to forming stable R-loops with hairpin structures forming on the displaced single-stranded DNA (S-loops).

DNA polymerase zeta can efficiently replicate structures formed by AT/TA repeat sequences and prevent their deletion.

Long AT repeat tracts form non-B DNA structures that stall DNA replication and cause chromosomal breakage. AT repeats are abundant in human common fragile sites (CFSs), genomic regions that undergo breakage under replication stress. Using an in vivo yeast model system containing AT-rich repetitive elements from human CFS FRA16D, we find that DNA polymerase zeta (Pol ζ) is required to prevent breakage and subsequent deletions at hairpin and cruciform forming (AT/TA)n sequences, with little to no role at an (A/T)28 repeat or a control non-structure forming sequence.

repeat expansion creates the unstable folate-sensitive fragile site FRA9A.

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and , is linked to multiple diseases. (GGGGCC)n expansions (Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immunostimulatory or damaged DNA is unknown.

expansion creates the unstable folate-sensitive fragile site FRA9A.

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and is linked to multiple diseases. (GGGGCC)n expansions ( Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown.

The DNA Replication Checkpoint Targets the Kinetochore for Relocation of Collapsed Forks to the Nuclear Periphery.

Unlabelled: Hairpin forming expanded CAG/CTG repeats pose significant challenges to DNA replication which can lead to replication fork collapse. Long CAG/CTG repeat tracts relocate to the nuclear pore complex to maintain their integrity. Forks impeded by DNA structures are known to activate the DNA damage checkpoint, thus we asked whether checkpoint proteins play a role in relocation of collapsed forks to the nuclear periphery in .

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks.

Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG) or (CTG) repeat tract.

What repeat expansion disorders can teach us about the Central Dogma.

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions.

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO (suppressors of transcriptional defects of hpr1Δ by overexpression) complex, and Trf4, a key component of the TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex involved in nuclear RNA polyadenylation and degradation, are necessary to prevent CAG fragility and repeat contractions in a Saccharomyces cerevisiae model system.

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.

Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties.

Homologous recombination within repetitive DNA.

Many microsatellite DNA sequences are able to form non-B form DNA secondary structures, such as hairpin loops, cruciforms, triplex DNA or G-quadruplexes. These DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks, which can be repaired by homologous recombination (HR). Recent work understanding HR at structure-forming repeats has focused on genetic requirements for replication fork restart, break induced replication (BIR) at broken forks, recombination during and after relocalization of breaks or stalled forks to the nuclear periphery, and how repair pathway choice and kinetics are navigated in the presence of a repeat tract.

Structure-forming repeats and their impact on genome stability.

Repetitive sequences throughout the genome are a major source of endogenous DNA damage, due to the propensity of many of them to form alternative non-B DNA structures that can interfere with replication, transcription, and DNA repair. These repetitive sequences are prone to breakage (fragility) and instability (changes in repeat number). Repeat fragility and expansions are linked to several diseases, including many cancers and neurodegenerative diseases, hence the importance of understanding the mechanisms that cause genome instability and contribute to these diseases.

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal.

Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR).

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear.

A Timeless Tale: G4 structure recognition by the fork protection complex triggers unwinding by DDX11 helicase.

How the replisome senses and deals with DNA secondary structures has been a mystery. A new study from the Sale and Pellegrini laboratories finds that the Timeless protein has a G-quadruplex binding domain that works together with the DDX11 helicase to facilitate replication of G4 DNA structures.

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities.

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast.

Location, Location, Location: The Role of Nuclear Positioning in the Repair of Collapsed Forks and Protection of Genome Stability.

Components of the nuclear pore complex (NPC) have been shown to play a crucial role in protecting against replication stress, and recovery from some types of stalled or collapsed replication forks requires movement of the DNA to the NPC in order to maintain genome stability. The role that nuclear positioning has on DNA repair has been investigated in several systems that inhibit normal replication. These include structure forming sequences (expanded CAG repeats), protein mediated stalls (replication fork barriers (RFBs)), stalls within the telomere sequence, and the use of drugs known to stall or collapse replication forks (HU + MMS or aphidicolin).

Relocation of Collapsed Forks to the Nuclear Pore Complex Depends on Sumoylation of DNA Repair Proteins and Permits Rad51 Association.

Expanded CAG repeats form stem-loop secondary structures that lead to fork stalling and collapse. Previous work has shown that these collapsed forks relocalize to nuclear pore complexes (NPCs) in late S phase in a manner dependent on replication, the nucleoporin Nup84, and the Slx5 protein, which prevents repeat fragility and instability. Here, we show that binding of the Smc5/6 complex to the collapsed fork triggers Mms21-dependent sumoylation of fork-associated DNA repair proteins, and that RPA, Rad52, and Rad59 are the key sumoylation targets that mediate relocation.

The nuclear pore complex prevents sister chromatid recombination during replicative senescence.

The Nuclear Pore Complex (NPC) has emerged as an important hub for processing various types of DNA damage. Here, we uncover that fusing a DNA binding domain to the NPC basket protein Nup1 reduces telomere relocalization to nuclear pores early after telomerase inactivation. This Nup1 modification also impairs the relocalization to the NPC of expanded CAG/CTG triplet repeats.

Distinct roles for H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126.

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in . Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.

Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae.

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.

Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D.

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication.

The role of fork stalling and DNA structures in causing chromosome fragility.

Alternative non-B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single-stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT-rich and break under replication stress, yet the molecular basis for their fragility is still being investigated.

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.