RESUMEN
Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Ácido Fólico/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Mitosis , Recombinasa Rad51/metabolismo , ADN/genética , ADN/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/genética , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/fisiopatología , Humanos , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Recombinasas/genética , Recombinasas/metabolismoRESUMEN
Particle image velocimetry is an important optical flow diagnostic tool due to its capacity for investigating a whole flow field without introducing disturbances. However, a significant drawback of PIV methods is their requirement for optical access, making capturing data in closed cavities and confined spaces extremely challenging. A potential approach to overcome this difficulty is miniaturising the system and placing the optical components inside the model. Conventional cross-correlation PIV methods do not allow this due to the size of current PIV cameras. In this study, a miniaturised autocorrelation-based stereo PIV system, which is volumetrically 1.2% of the conventional PIV cameras, was developed and tested. The miniature system is compared with a conventional stereo PIV in wind tunnel experiments up to 16 m/s free stream velocity and a 1.6% velocity difference is observed in the boundary layer flow. Despite a comparatively slow measurement rate of 4.5 Hz, the miniature PIV system demonstrates the ability to measure inside confined spaces and cavities and the ability to be mounted on board models and vehicles. However, limitations remain around conducting measurements with large velocity ranges and with regions of reversed flow due to the challenge of resolving a velocity of 0 m/s.
RESUMEN
Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.
Asunto(s)
RecQ Helicasas/química , Adenosina Difosfato/química , Cristalografía por Rayos X , ADN/química , Modelos Moleculares , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Anticuerpos de Dominio Único/química , Zinc/químicaRESUMEN
Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/metabolismo , G-Cuádruplex , Genoma Humano/genética , Mitosis , Animales , Línea Celular , ADN Helicasas/química , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Inestabilidad Genómica , Humanos , Inmunoprecipitación , Ratones , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Conformación de Ácido Nucleico , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismoRESUMEN
Oncogene activation during tumour development leads to changes in the DNA replication programme that enhance DNA replication stress. Certain regions of the human genome, such as common fragile sites and telomeres, are particularly sensitive to DNA replication stress due to their inherently 'difficult-to-replicate' nature. Indeed, it appears that these regions sometimes fail to complete DNA replication within the period of interphase when cells are exposed to DNA replication stress. Under these conditions, cells use a salvage pathway, termed 'mitotic DNA repair synthesis (MiDAS)', to complete DNA synthesis in the early stages of mitosis. If MiDAS fails, the ensuing mitotic errors threaten genome integrity and cell viability. Recent studies have provided an insight into how MiDAS helps cells to counteract DNA replication stress. However, our understanding of the molecular mechanisms and regulation of MiDAS remain poorly defined. Here, we provide an overview of how DNA replication stress triggers MiDAS, with an emphasis on how common fragile sites and telomeres are maintained. Furthermore, we discuss how a better understanding of MiDAS might reveal novel strategies to target cancer cells that maintain viability in the face of chronic oncogene-induced DNA replication stress.
Asunto(s)
Sitios Frágiles del Cromosoma , Replicación del ADN/fisiología , Homeostasis del Telómero/fisiología , Reparación del ADN , Inestabilidad Genómica , Humanos , Neoplasias/genéticaRESUMEN
Telomeres resemble common fragile sites (CFSs) in that they are difficult-to-replicate and exhibit fragility in mitosis in response to DNA replication stress. At CFSs, this fragility is associated with a delay in the completion of DNA replication until early mitosis, whereupon cells are proposed to switch to a RAD52-dependent form of break-induced replication. Here, we show that this mitotic DNA synthesis (MiDAS) is also a feature of human telomeres. Telomeric MiDAS is not restricted to those telomeres displaying overt fragility, and is a feature of a wide range of cell lines irrespective of whether their telomeres are maintained by telomerase or by the alternative lengthening of telomeres (ALT) mechanism. MiDAS at telomeres requires RAD52, and is mechanistically similar to CFS-associated MiDAS, with the notable exception that telomeric MiDAS does not require the MUS81-EME1 endonuclease. We propose a model whereby replication stress initiates a RAD52-dependent form of break-induced replication that bypasses a requirement for MUS81-EME1 to complete DNA synthesis in mitosis.
RESUMEN
A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all individuals) or rare (RFSs; present in only a few individuals). Interest in fragile sites has remained high since their discovery in 1965, because of their association with human disease. CFSs are recognized as drivers of oncogene activation and genome instability in cancer cells, while some RFSs are associated with neurodegenerative diseases. This review summaries our current understanding of the nature and causes of fragile site "expression", including the recently characterized phenomenon of telomere fragility. In particular, we focus on a description of the methodologies and technologies for detection and analysis of chromosome fragile sites.
Asunto(s)
Sitios Frágiles del Cromosoma , Inestabilidad Genómica , Hibridación Fluorescente in Situ , Animales , Replicación del ADN , Expresión Génica , Sitios Genéticos , Humanos , Hibridación Fluorescente in Situ/métodosRESUMEN
Our conventional understanding of the process of DNA replication is that it occurs in the S-phase of the cell division cycle. However, during investigations into the mechanism by which common fragile sites (CFSs) drive genome instability, we observed that some DNA synthesis was still occurring in early mitosis at these loci. This curious phenomenon of mitotic DNA synthesis (which we now term "MiDAS") appears to be a form of break-induced DNA replication (BIR), a DNA repair process based on homologous recombination that has been characterized in detail only in lower eukaryotes. During MiDAS, it is proposed that parts of the human genome that are not fully replicated when cells enter mitotic prophase complete their replicative cycle at that point. To date, the loci that most depend upon this process are those whose replication can be affected by oncogene-induced DNA replication stress (RS), most notably, CFSs. From our studies, it is clear that the successful completion of MiDAS at CFSs can minimize chromosome missegregation and nondisjunction. Nevertheless, it is still not clear which loci that can undergo MiDAS, whether MiDAS is associated with mutations or genome rearrangements, or whether MiDAS really is a form of BIR. In this review, we describe methods for detecting MiDAS both in prometaphase cells and directly on isolated metaphase chromosomes. In addition, we have included methods for combining MiDAS detection either with immunofluorescence (IF) detection of proteins that are recruited to the MiDAS loci, or with fluorescence in situ hybridization using probes that target specific genomic loci.