The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.
- MeSH
- časové faktory MeSH
- chromozomální nestabilita MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- DNA biosyntéza genetika MeSH
- endodeoxyribonukleasy metabolismus MeSH
- endonukleasy genetika metabolismus MeSH
- fosforylace MeSH
- fragilní místa na chromozomu * MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- helikasy RecQ genetika metabolismus MeSH
- lidé MeSH
- mitóza * MeSH
- oprava DNA * MeSH
- poškození DNA MeSH
- rekombinasa Rad51 metabolismus MeSH
- replikační počátek * MeSH
- RNA interference MeSH
- segregace chromozomů MeSH
- transfekce MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.
- MeSH
- chromatografie afinitní MeSH
- DNA vazebné proteiny fyziologie MeSH
- fragilní místa na chromozomu MeSH
- kontrolní body buněčného cyklu MeSH
- lidé MeSH
- oprava DNA fyziologie MeSH
- proteomika metody MeSH
- replikace DNA fyziologie MeSH
- xeroderma pigmentosum MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.
- MeSH
- chromatin genetika MeSH
- chromozomální proteiny, nehistonové metabolismus účinky záření MeSH
- DNA vazebné proteiny účinky záření MeSH
- fibroblasty účinky záření MeSH
- fragilní místa na chromozomu genetika MeSH
- histony účinky záření MeSH
- jaderné proteiny metabolismus účinky záření MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny metabolismus účinky záření MeSH
- nestabilita genomu MeSH
- oprava DNA genetika MeSH
- osteosarkom MeSH
- poškození DNA účinky záření MeSH
- ribozomy genetika MeSH
- transkripční faktory metabolismus účinky záření MeSH
- záření gama MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This work is a continuation of our effort to determine the structure responsible for expansion of the (CGG)(n) motif that results in fragile X chromosome syndrome. In our previous report, we demonstrated that the structure adopted by an oligonucleotide with this repeat sequence is not a quadruplex as was suggested by others. Here we demonstrate that (CGG) runs adopt another anomalous arrangement-a left-handed Z-DNA structure. The Z-DNA formation was induced by high salt and millimolar concentrations of Ni(2+) ions and likelihood of its formation increased with increasing number of repeats. In an oligonucleotide in which the CGG runs were interrupted by AGG triplets, as is observed in genomes of healthy individuals, the hairpin conformation was stabilized and Z-DNA formation was hindered. We show here that methylation of the (CGG) runs markedly stabilized Z-DNA formation. We hypothesize that rather than in the expansion process the Z-DNA may be formed by long, expanded (CGG) stretches that become hypermethylated; this would inhibit transcription resulting in disease.
- MeSH
- cirkulární dichroismus MeSH
- fragilní místa na chromozomu genetika MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- lidské chromozomy X genetika MeSH
- sekvence nukleotidů MeSH
- syndrom fragilního X genetika MeSH
- trinukleotidové repetice MeSH
- Z-DNA chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Genomes are exposed to various external stimuli that induce DNA damage in the form of single- or double-stranded DNA breaks. Fragile sites in the human genome are sensitive to genotoxic stress and, when not appropriately repaired, are responsible for chromosomal aberrations, including the gene amplifications observed in a variety of tumors. Moreover, when DNA lesions from different chromosomes are in close proximity and not repaired, the probability of chromosome translocations is greatly increased. These events can be induced by ionizing radiation that, in a majority of cells, induces a G2/M cell cycle arrest and is characterized by the repositioning of many tumor-related genes closer to the nuclear interior. On the basis of this knowledge, we review functional and structural aspects of chromosomal rearrangements and the DNA repair machinery.
