Updating the mechanisms of common fragile site instability: how to reconcile the different views?

Common fragile sites (CFSs) are large chromosomal regions long identified by conventional cytogenetics as sequences prone to breakage in cells subjected to replication stress. The interest in CFSs came from their key role in the formation of DNA damage, resulting in chromosomal rearrangements. The instability of CFSs was notably correlated with the appearance of genome instability in precancerous lesions and during tumor progression. Identification of the molecular mechanisms responsible for their instability therefore represents a major challenge. A number of data show that breaks result from mitotic entry before replication completion but the mechanisms responsible for such delayed replication of CFSs and relaxed checkpoint surveillance are still debated. In addition, clues to the molecular events leading to breakage just start to emerge. We present here the results of recent reports addressing these questions

Stepwise Activation of the ATR Signaling Pathway upon Increasing Replication Stress Impacts Fragile Site Integrity

<div><p>Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.</p></div

Multiple roles for kinases in DNA replication

DNA replication is carried out by the replisome, which includes several proteins that are targets of cell-cycle-regulated kinases. The phosphorylation of proteins such as replication protein A, DNA polymerase-α and -δ, replication factor C, flap endonuclease 1 and DNA ligase I leads to their inactivation, suggesting that phosphorylation is important in the prevention of re-replication. Moreover, the phosphorylation of several of these replication proteins has been shown to block their association with the 'moving platform'—proliferating cell nuclear antigen. Therefore, phosphorylation seems to be a crucial regulator of replisome assembly and DNA replication, although its precise role in these processes remains to be clarified

Palbociclib interferes with replication origin firing in a pRb independent manner

Abstract Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, promoting needs to understand more about the mechanism of inhibitors beyond pRb. Our study demonstrates here how palbociclib impairs the origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA without any induced cell cycle arrest. Furthermore, palbociclib treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of proteins of the Pre-initiation complex (Pre-IC) on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and on its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib to target genomic instability towards pRb deficient patients. Significance Statement Palbociclib is a promising anticancer drug for pRb-proficient cell, particularly for hormonal receptor positive breast cancer, that induces the cell cycle arrest. But what about pRb deficient cell lines ? Our results show that Palbociclib disturb the DNA replication process inducing a replicative stress, an increase of DNA damages and leading to a significant decrease in cell viability. Palbociclib impairs the DNA synthesis reducing the number of active origins with the decrease of availability of the pre-initiation complexes. We believe that the demonstration of this effect of palbociclib on pRb-deficient cells may be a new therapeutic entry point in combination with other treatments for these types of cancer. Replicative stress can be one of weaknesses of pRb defficient cancer cells

Moderate fork slowing is not associated with formation of ssDNA foci.

<p>(A) ssDNA detection scheme. Cells hemi-substituted throughout their genome after growth in the presence of CldU for about 1.5 cell generations were treated or not with aphidicolin or HU for different periods of time prior to fixation. CldU was immuno-detected without DNA denaturation, which only permits visualization of substituted and single-stranded regions. (B) Co-detection of ssDNA (red) and chromatin bound PCNA (green) by immunostaining of untreated cells (-) and cells treated as indicated. Nuclei were counterstained with DAPI. (C) Typical pattern of PCNA and CldU foci in cells treated with HU 1 mM during 1 h. (D) Typical pattern of PCNA and CldU foci in cells depleted of ATR and treated with 0.3 µM of aphidicolin for 4 h.</p

The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen

The coordination of chromatin assembly with DNA replication, which is essential for genomic stability, requires the combined activation of histone deposition with the firing of replication origins. We report here the direct interaction of chromatin assembly factor 1 (CAF1), a key factor involved in histone deposition, with the replication kinase Cdc7-Dbf4. We isolated a complex containing both the largest subunit of CAF1 (p150) and the Cdc7-Dbf4 kinase specifically in S phase and thus prove the existence of this interaction in vivo. We then show that the Cdc7-Dbf4 kinase efficiently phosphorylates p150. This event induces a change in p150 oligomerization state, which promotes binding to proliferating cell nuclear antigen (PCNA). Conversely, CAF1 recruitment is reduced in a PCNA/DNA loading assay using Cdc7-depleted extracts. Our data define p150 as a new target for this kinase with implications for the coordination between DNA replication and CAF1 functions

Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage

International audienceMammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates

ATR-depletion enhances formation of ssDNA foci and fork asymmetry.

<p>(A) Percentage of S-phase cells with ssDNA foci in the indicated conditions of transfection and aphidicolin treatment. Mean ± s.e.m are presented. (B) Upper panel: Western blot analyses of total cell extracts 48 h post-transfection with a NONsi RNA or siRNAs specific to ATR or/and Mre11. Loading control: β-actin. Lower panel: percentage S-phase cells (mean ± s.e.m) displaying ssDNA foci in populations of cells depleted of ATR and treated or not with aphidicolin 0.3 µM. Mre11 status is indicated (siMre11 or treatment with Mirin: 100 µM). (C) Percentage of FISH signal co-localizing with ssDNA foci in cells depleted of ATR and treated with 0.3 µM aphidicolin. The immunostaining of ssDNA and PCNA was combined with FISH with BAC probes. Upper panel: JEFF lymphoblastoid cells, the BACs probe early replicating regions (875H7 and 456N14) and late replicating regions (321A23, 660J14, and 641C17 that corresponds to <i>FRA3B</i>, the major CFS in lymphocytes). Lower panel: MRC-5 fibroblasts, BAC 456N14 probe for an early replicating region, other BACs correspond to late replicating regions (875H7, 321A23, 641C17, and 660J14 which corresponds to the major CFS in fibroblast mapping at 3q13.3). (D) Distributions of fork asymmetry in cells transfected and treated as indicated. Forks travelling in the <i>FRA3B</i> locus are represented by red circles (<i>n</i> = 17) and in the bulk genome by black circles (untreated: NONsi, <i>n</i> = 128; siCHk1, <i>n</i> = 103; siATR, <i>n</i> = 147; with aphidicolin: NONsi, <i>n</i> = 121; siCHk1, <i>n</i> = 132; siATR, <i>n</i> = 123). Horizontal orange lines represent the medians of fork distributions. Medians and <i>P</i> values are indicated above the distributions (not significant, <i>ns</i>).</p

Moderate fork slowing triggers neither γ-H2AX foci nor ATM activation.

<p>(A) Western blot analysis of ATM-Ser1981 phosphorylation and γ-H2AX in exponentially growing cells, untreated (-) or treated as indicated. (B) Immunostaining of γ-H2AX together with chromatin-bound PCNA in untreated cells (-) and in cells treated with aphidicolin 0.6 µM for the indicated periods of time, or for 1 h with HU 1 mM.</p

The RBBP6/ZBTB38/MCM10 Axis Regulates DNA Replication and Common Fragile Site Stability

Summary: Faithful DNA replication is essential for the maintenance of genome integrity. Incomplete genome replication leads to DNA breaks and chromosomal rearrangements, which are causal factors in cancer and other human diseases. Despite their importance, the molecular mechanisms that control human genome stability are incompletely understood. Here, we report a pathway that is required for human genome replication and stability. This pathway has three components: an E3 ubiquitin ligase, a transcriptional repressor, and a replication protein. The E3 ubiquitin ligase RBBP6 ubiquitinates and destabilizes the transcriptional repressor ZBTB38. This repressor negatively regulates transcription and levels of the MCM10 replication factor on chromatin. Cells lacking RBBP6 experience reduced replication fork progression and increased damage at common fragile sites due to ZBTB38 accumulation and MCM10 downregulation. Our results uncover a pathway that ensures genome-wide DNA replication and chromosomal stability. : DNA replication duplicates the genome at every cell cycle. Miotto et al. have now identified a molecular pathway that ensures proper replication of the mammalian genome. Common fragile sites are regions in the mammalian genome that are prone to loss when DNA replication is suboptimal. The new data show that common fragile sites are exquisitely sensitive to the activity of this RBBP6/ZBTB38/MCM10 axis, suggesting that deregulation of these factors may underlie genome instability and human disease