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And RAD51-Dependent Mitotic DNA Synthesis at the Fragile X Locus in Human Cells

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And RAD51-Dependent Mitotic DNA Synthesis at the Fragile X Locus in Human Cells Folate stress induces SLX1- and RAD51-dependent mitotic DNA synthesis at the fragile X locus in human cells Lorenza Garribbaa,1,2, Victoria A. Bjerregaarda,1,3, Marisa M. Gonçalves Dinisa, Özgün Özera,4, Wei Wua, Despoina Sakellarioub,5, Javier Pena-Diazb, Ian D. Hicksona,b, and Ying Liua,b,6 aCenter for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark; and bCenter for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark Edited by Lorraine S. Symington, Columbia University Iriving Medical Center, New York, NY, and accepted by Editorial Board Member Philip C. Hanawalt May 15, 2020 (received for review December 3, 2019) Folate deprivation drives the instability of a group of rare fragile than 55 CGG repeats, while the mutant form of this locus has over sites (RFSs) characterized by CGG trinucleotide repeat (TNR) 200 repeats. Abnormal expansion of this TNR can lead to hyper- sequences. Pathological expansion of the TNR within the FRAXA methylation and epigenetic silencing of FMR1, which leads to the locus perturbs DNA replication and is the major causative factor development of FXS (10). To our knowledge, 24 folate-sensitive for fragile X syndrome, a sex-linked disorder associated with cog- RFSs have been identified (5) but only 6 of them (including nitive impairment. Although folate-sensitive RFSs share many fea- FRAXA) have been characterized for their precise genomic tures with common fragile sites (CFSs; which are found in all location (11). individuals), they are induced by different stresses and share no RFSs appear on metaphase chromosomes as gaps or breaks in sequence similarity. It is known that a pathway (termed MiDAS) is otherwise fully condensed chromatin (called RFS “expression”). employed to complete the replication of CFSs in early mitosis. This Much of our understanding of the underlying cause of chromo- process requires RAD52 and is implicated in generating transloca- some fragile site expression has been derived from studies of tions and copy number changes at CFSs in cancers. However, it is another class of fragile sites that exist in all individuals, known as unclear whether RFSs also utilize MiDAS and to what extent the common fragile sites (CFSs). It is generally considered that CFS MEDICAL SCIENCES fragility of CFSs and RFSs arises by shared or distinct mechanisms. FRAXA Here, we demonstrate that MiDAS does occur at following Significance folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on Folate deficiency is associated with multiple disorders in hu- RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA mans. Through the analysis of the fragile X syndrome locus leads to severe locus instability and missegregation in mitosis. We (FRAXA) in immortalized human lymphocytes or fibroblasts, propose that break-induced DNA replication is required for the we demonstrate that FRAXA undergoes DNA synthesis in mi- replication of FRAXA under folate stress and define a cellular func- tosis (MiDAS). We demonstrate that this process occurs via tion for human SLX1. These findings provide insights into how break-induced DNA replication and requires the SLX1/SLX4 folate deprivation drives instability in the human genome. endonuclease complex, the RAD51 recombinase and POLD3, a subunit of polymerase delta. We also demonstrate that other MiDAS | structure-specific endonucleases | chromosome fragile sites | loci undergo MiDAS upon folate stress. This study reveals a homologous recombination | break-induced DNA replication (BIR) function of human SLX1 in the maintenance of FRAXA stability and provides evidence that, in addition to FRAXA, MiDAS oc- olate is a B type vitamin that functions as a carrier for one- curs at other loci following folate deprivation. These findings Fcarbon units, which are essential for DNA and RNA syn- provide insight into the diverse and detrimental consequences thesis. Humans cannot synthesize folate and, therefore, rely on of folate deficiency in human cells. dietary sources of this nutrient. In human populations in which folic acid supplementation is absent, folate deficiency is observed Author contributions: L.G., V.A.B., I.D.H., and Y.L. designed research; L.G., V.A.B., frequently (1–4). Because of the requirement for folate in the M.M.G.D., Ö.Ö., W.W., D.S., and J.P.-D. performed research; L.G., V.A.B., Ö.Ö., W.W., synthesis of nucleotides, folate deficiency can destabilize the and Y.L. contributed new reagents/analytic tools; L.G., V.A.B., M.M.G.D., I.D.H., and Y.L. analyzed data; and L.G., I.D.H., and Y.L. wrote the paper. human genome through influencing the fidelity of DNA repli- cation. In particular, it is established that a subgroup of so-called The authors declare no competing interest. rare fragile sites (RFSs), which are found in less than 5% of the This article is a PNAS Direct Submission. L.S.S. is a guest editor invited by the Editorial Board. human population, are highly sensitive to folate deprivation. This open access article is distributed under Creative Commons Attribution-NonCommercial- These folate-sensitive RFSs generally encompass CGG tri- NoDerivatives License 4.0 (CC BY-NC-ND). nucleotide repeat sequences, which are prone to expand in length 1L.G. and V.A.B. contributed equally to this work. via a mechanism that remains to be fully elucidated. Most in- 2Present address: Genome Integrity, Department of Experimental Oncology, European triguingly, when these CGG repeats expand beyond a certain length, Institute of Oncology, 20139 Milan, Italy. the locus exhibits fragility in metaphase when cells are challenged 3 “ ” Present address: Department of Clinical Genetics, Kennedy Center, Copenhagen Univer- with folate stress conditions, such as when cells are deprived of sity Hospital, Rigshospitalet, 2600 Glostrup, Denmark. folate or exposed to the thymidylate synthase inhibitor, fluo- 4Present address: Cell Division, The Institute of Cancer Research, London SW3 6JB, rodeoxyuridine (FdU) (5). It is well-established that, when the copy United Kingdom. number of the TNR sequences expands beyond a critical size, the 5Present address: Genome Integrity, Danish Cancer Society Research Center, DK-2100 development of specific neurological diseases such as fragile X Copenhagen, Denmark. syndrome (FXS) can be triggered (6–9). The genomic locus asso- 6To whom correspondence may be addressed. Email: [email protected]. ciated with FXS, FRAXA, contains unstable CGG repeats located at This article contains supporting information online at https://www.pnas.org/lookup/suppl/ the 5′-untranslated region (5′-UTR) of the FMR1 gene. In the doi:10.1073/pnas.1921219117/-/DCSupplemental. general population, the FRAXA-associated TNR consists of less First published June 29, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1921219117 PNAS | July 14, 2020 | vol. 117 | no. 28 | 16527–16536 Downloaded by guest on September 24, 2021 expression results from a localized failure to undergo adequate mutation allele. In addition, analyses requiring small interfering compaction of the locus during early mitosis due to incomplete RNA (siRNA)-mediated protein depletion were performed on DNA replication during interphase (12). CFSs are recognized as an hTERT-immortalized fibroblast cell line (GM05848) that has being a key driver of genome instability in cancer cells (13, 14). a full mutation FRAXA allele (SI Appendix, Fig. S1). In cultured cells, the fragility of CFSs can be strongly enhanced if We first analyzed whether folate stress can induce MiDAS at the cells experience DNA replication stress, such as traversing S the FRAXA locus or elsewhere in the human genome. For this, phase in the presence of a low dose of the DNA polymerase we treated GM06865, GM06891, and GM09237 cells with 0.5 μM inhibitor, aphidicolin (APH). In human cancers, however, this FdU for 17 h, arrested them at the G2/M phase boundary with the stress is widespread and intrinsic, because it is driven by the action of CDK1 inhibitor, RO3306, and then released the cells into mitosis in activated oncogenes. In response to replication stress, human cells the presence of the thymidine analog, EdU, to mark sites of new activate a signaling pathway that leads to the localization of DNA DNA synthesis. EdU detection was then performed on metaphase repair proteins, including the SLX4-associated, structure-specific chromosome spreads (18, 30) (Fig. 1A). We observed that a small endonuclease (SSE), MUS81-EME1 to CFS loci (15–17). This, in number of EdU foci (typically up to three) could be detected in all turn, facilitates the completion of DNA replication at CFSs only cell lines in the absence of folate stress, but that exposure to FdU after cells have entered early mitosis, using a form of unscheduled induced a significantly higher number of EdU foci (Fig. 1 B and C). DNA synthesis known as “MiDAS” (mitotic DNA synthesis) (18, This suggested that several loci in the human genome can be af- 19). Mechanistically, MiDAS likely represents a form of break- fected by folate stress. Interestingly, the vast majority (73–94% on induced DNA replication (BIR), which is a subpathway of homol- average depending on the cell line) of the EdU foci detected were ogous recombination repair acting at sites of broken/collapsed DNA located on only one sister-chromatid (Fig. 1 B and D), which is a replication forks (20). BIR has largely been characterized in budding much higher percentage than that observed at CFSs in cells treated yeast, where it has been shown to be a conservative form of DNA with APH (19). These data indicate that the majority of folate synthesis dependent on the Rad51 recombinase and Pol32 (21, 22). stress-induced MiDAS events occur via a conservative form of At CFSs, MiDAS is apparently triggered during the onset of chro- DNA synthesis, which has been proposed previously to be mediated matin condensation in the mitotic prophase and is facilitated not via BIR (18, 19).
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