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Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218


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.

Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
Nucleic Acids Res ; 47(15): 8004-8018, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31180492


Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.

Sítios Frágeis do Cromossomo , Instabilidade Genômica , Proteoma/metabolismo , Proteômica/métodos , Proteína Nuclear Ligada ao X/metabolismo , Quebra Cromossômica , Reparo do DNA , Replicação do DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteoma/genética , Interferência de RNA , Espectrometria de Massas em Tandem , Proteína Nuclear Ligada ao X/genética
Proc Natl Acad Sci U S A ; 115(51): 13003-13008, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30509972


The instability of chromosome fragile sites is implicated as a causative factor in several human diseases, including cancer [for common fragile sites (CFSs)] and neurological disorders [for rare fragile sites (RFSs)]. Previous studies have indicated that problems arising during DNA replication are the underlying source of this instability. Although the role of replication stress in promoting instability at CFSs is well documented, much less is known about how the fragility of RFSs arises. Many RFSs, as exemplified by expansion of a CGG trinucleotide repeat sequence in the fragile X syndrome-associated FRAXA locus, exhibit fragility in response to folate deficiency or other forms of "folate stress." We hypothesized that such folate stress, through disturbing the replication program within the pathologically expanded repeats within FRAXA, would lead to mitotic abnormalities that exacerbate locus instability. Here, we show that folate stress leads to a dramatic increase in missegregation of FRAXA coupled with the formation of single-stranded DNA bridges in anaphase and micronuclei that contain the FRAXA locus. Moreover, chromosome X aneuploidy is seen when these cells are exposed to folate deficiency for an extended period. We propose that problematic FRAXA replication during interphase leads to a failure to disjoin the sister chromatids during anaphase. This generates further instability not only at FRAXA itself but also of chromosome X. These data have wider implications for the effects of folate deficiency on chromosome instability in human cells.

Sítios Frágeis do Cromossomo , Cromossomos Humanos X , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Linfócitos/patologia , Mitose , Estresse Fisiológico , Células Cultivadas , Replicação do DNA , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Linfócitos/metabolismo , Masculino , Expansão das Repetições de Trinucleotídeos
Methods Enzymol ; 601: 45-58, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29523241


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.

Técnicas Genéticas , Mitose , Reparo de DNA por Recombinação , Linhagem Celular , DNA/metabolismo , Humanos , Metáfase , Prometáfase
Oncotarget ; 8(23): 36996-37008, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28445142


Oncogene activation is an established driver of tumorigenesis. An apparently inevitable consequence of oncogene activation is the generation of DNA replication stress (RS), a feature common to most cancer cells. RS, in turn, is a causal factor in the development of chromosome instability (CIN), a near universal feature of solid tumors. It is likely that CIN and RS are mutually reinforcing drivers that not only accelerate tumorigenesis, but also permit cancer cells to adapt to diverse and hostile environments. This article reviews the genetic changes present in cancer cells that influence oncogene-induced RS and CIN, with a particular emphasis on regions of the human genome that show enhanced sensitivity to the destabilizing effects of RS, such as common fragile sites. Because RS exists in a wide range of cancer types, we propose that the proteins involved counteracting this stress are potential biomarkers for indicating the degree of RS in cancer specimens. To test this hypothesis, we conducted a pilot study to validate whether some of proteins that are known from in vitro studies to play an essential role in the RS pathway could be suitable as a biomarker. Our results indicated that this is possible. With this review and pilot study, we aim to accelerate the development of a biomarker for analysis of RS in tumor biopsy specimens, which could ultimately help to stratify patients for different forms of therapy such as the RS inhibitors already undergoing clinical trials.

Biomarcadores Tumorais/genética , Instabilidade Cromossômica , Replicação do DNA , Neoplasias/genética , Carcinogênese/genética , Genoma Humano/genética , Humanos , Mutação , Neoplasias/patologia , Projetos Piloto