Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism

Özer, Özgün; Bhowmick, Rahul; Liu, Ying; Hickson, Ian D.

doi:10.18632/oncotarget.24745

Cited by 80 publications

(90 citation statements)

References 26 publications

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“…These findings suggest that a temporal separation between DNA synthesis and chromosome segregation does not seem to be a strict rule for eukaryotic cells. However, DNA synthesis outside S phase is particularly prevalent in cells exhibiting aneuploidy (58,59), suggesting it may be less effective at maintaining genome integrity. Here, we provide evidence that L. major, a eukaryotic organism with constitutive mosaic aneuploidy (at least in parasite cells derived from, or proliferating in the insect vector) (38,60), achieves full genome duplication using DNA synthesis outside S phase, including G2/M and G1.…”

Section: Discussionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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Once every cell cycle, DNA replication takes place to allow cells to duplicate their genome and segregate the two resulting copies into offspring cells. In eukaryotes, the number of DNA replication initiation loci, termed origins, is proportional to chromosome size. However, previous studies have suggested that in Leishmania, a group of single-celled eukaryotic parasites, DNA replication starts from just a single origin per chromosome, which is predicted to be insufficient to secure complete genome duplication within S phase.Here, we show that the paucity of origins activated in early S phase is balanced by DNA synthesis activity outside S phase. Simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1 is exclusively found at the origins used in early S phase, while subtelomeric DNA replication can only be linked to AcH3 and displays persistent activity through the cell cycle, including in G2/M and G1 phases. We also show that subtelomeric DNA replication, unlike replication from the previously mapped origins, is sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex.Our work indicates that Leishmania genome transmission relies on an unconventional DNA replication programme, which may have implications for genome stability in this important parasite. sequence read depth in the former relative to the latter. As result, DNA synthesis very late in S-phase, or that occurs outside of S-phase, would escape detection. To test this, we modified the MFA-seq approach in order that DNA content enrichment could be calculated in replicating cells relative to naturally occurring non-replicative cells. Mapping origins of replication by calculating DNA enrichment in exponentially growing cells versus cells in a stationary state has been used successfully in bacteria (42) and yeast (43). Thus, we reasoned that L. major in stationary phase could also serve as a non-replicative control for normalization in MFA-seq analysis compared with cells that are growing exponentially. To test this, we used flow cytometry to compare DNA content of exponentially growing and stationary phase cells and, in addition, compared the capacity of the two cell populations to incorporate the thymidine analogue 5-ethynyl-2'deoxyuridine (EdU; Fig. 1A,B). Flow cytometry showed that the proportion of cells in S phase (with DNA content between 1C and 2C) was substantially lower in a population of stationary cells compared with exponentially growing cells (Fig. 1A). Concomitantly, stationary phase cells, unlike exponentially growing cells, failed to incorporate EdU, even upon relatively long periods of incubation ( Fig. 1B). Thus, L. major cells in stationary phase do not perform any detectable DNA synthesis and were therefore deemed suitable to be used as the non-replicative sample in MFA-seq analysis.Next, we compared MFA-seq profiles using read depth ratios from FACS-sorted early S (ES) and G2 cells, and from exponentially growing cells (EXP) and stationary (STA) cells (Fig. 1C). As reported ...

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Section: Discussionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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“…APBs have been proposed to play a critical role in ALT by clustering telomeres and DNA repair factors together, thus concentrating substrates and enzymes required for recombination-based telomere elongation (Henson et al 2002;Cesare and Reddel 2010;Chung et al 2011;Chung et al 2012;Min et al 2019;Verma et al 2019). In agreement with this hypothesis, it has recently been shown that telomeres can be elongated during mitosis in APB-like foci in a process termed mitotic DNA synthesis MiDAS (Ozer et al 2018;Min et al 2019). Moreover, depletion of PML by siRNA has been shown to reduce telomere elongation (Osterwald et al 2015).…”

Section: Introductionmentioning

confidence: 90%

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres

Loe

Zhang

et al. 2020

Preprint

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Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by utilizing the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed Alternative Lengthening of Telomeres (ALT). Cells that employ ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML-Bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We find that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

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“…When we treated Pol η-depleted ALT cells with low dose of aphidicolin followed by EdU pulse, EdU positive newly synthesized DNA was observed on telomeric sequences of metaphase chromosomes, demonstrating that MiDAS is also a feature of human telomeres [24] ( Figure 1C). In fact, a recent work reported that telomeric MiDAS seems also to occur in cells maintaining their telomeres by the telomerase [29].…”

Section: Why the Cells Use Midas?mentioning

confidence: 99%

“…Therefore, RAD52 appears to hold a cardinal function at an SLX4-dependent step in CFS-associated MiDAS prior to the involvement of MUS81 or POLD3 (see model in Figure 2). At telomeres, MiDAS resembles to CFS-related MiDAS as it requires also RAD52 [29] but it does not require the MUS81-EME1 endonuclease, suggesting that another nuclease might be involved or MiDAS events do not all require an initial endonuclease cut.…”

Section: Mechanisms and Molecular Actors Involved In Midasmentioning

confidence: 99%

When RAD52 Allows Mitosis to Accept Unscheduled DNA Synthesis

Franchet

Hoffmann

2019

Cancers

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Faithful duplication of the human genome during the S phase of cell cycle and accurate segregation of sister chromatids in mitosis are essential for the maintenance of chromosome stability from one generation of cells to the next. Cells that are copying their DNA in preparation for division can suffer from 'replication stress' (RS) due to various external or endogenous impediments that slow or stall replication forks. RS is a major cause of pathologies including cancer, premature ageing and other disorders associated with genomic instability. It particularly affects genomic loci where progression of replication forks is intrinsically slow or problematic, such as common fragile site (CFS), telomeres, and repetitive sequences. Although the eukaryotic cell cycle is conventionally thought of as several separate steps, each of which must be completed before the next one is initiated, it is now accepted that incompletely replicated chromosomal domains generated in S phase upon RS at these genomic loci can result in late DNA synthesis in G2/M. In 2013, during investigations into the mechanism by which the specialized DNA polymerase eta (Pol η) contributes to the replication and stability of CFS, we unveiled that indeed some DNA synthesis was still occurring in early mitosis at these loci. This surprising observation of mitotic DNA synthesis that differs fundamentally from canonical semi-conservative DNA replication in S-phase has been then confirmed, called "MiDAS"and believed to counteract potentially lethal chromosome mis-segregation and non-disjunction. While other contributions in this Special Issue of Cancers focus on the role of RAS52RAD52 during MiDAS, this review emphases on the discovery of MiDAS and its molecular effectors.

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Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism

Cited by 80 publications

References 26 publications

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres

When RAD52 Allows Mitosis to Accept Unscheduled DNA Synthesis

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