Specialized replication mechanisms maintain genome stability at human centromeres.

The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.

CENP-B-mediated DNA loops regulate activity and stability of human centromeres.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Enrichment of centromeric DNA from human cells.

Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, and this can be extremely costly. To bypass these issues, we have developed a technique, named CenRICH, to enrich for centromeric DNA from human cells based on selective restriction digestion and size fractionation. Combining restriction enzymes cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of fragments >20 kb, resulted in over 25-fold enrichment in centromeric DNA. High-throughput sequencing revealed that up to 60% of the DNA in the enriched samples is made of centromeric repeats. We show that this method can be used in combination with long-read sequencing to investigate the DNA methylation status of certain centromeres and, with a specific enzyme combination, also of their surrounding regions (mainly HSATII). Finally, we show that CenRICH facilitates single-molecule analysis of replicating centromeric fibers by DNA combing. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy.

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells.

Homologous recombination deficient (HR(-)) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR(-) cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR(-) cells. Reciprocally, supplying nucleotide precursors to HR(-) cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.

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.

DNA Replication Stress and Chromosomal Instability: Dangerous Liaisons.

Chromosomal instability (CIN) is associated with many human diseases, including neurodevelopmental or neurodegenerative conditions, age-related disorders and cancer, and is a key driver for disease initiation and progression. A major source of structural chromosome instability (s-CIN) leading to structural chromosome aberrations is "replication stress", a condition in which stalled or slowly progressing replication forks interfere with timely and error-free completion of the S phase. On the other hand, mitotic errors that result in chromosome mis-segregation are the cause of numerical chromosome instability (n-CIN) and aneuploidy. In this review, we will discuss recent evidence showing that these two forms of chromosomal instability can be mechanistically interlinked. We first summarize how replication stress causes structural and numerical CIN, focusing on mechanisms such as mitotic rescue of replication stress (MRRS) and centriole disengagement, which prevent or contribute to specific types of structural chromosome aberrations and segregation errors. We describe the main outcomes of segregation errors and how micronucleation and aneuploidy can be the key stimuli promoting inflammation, senescence, or chromothripsis. At the end, we discuss how CIN can reduce cellular fitness and may behave as an anticancer barrier in noncancerous cells or precancerous lesions, whereas it fuels genomic instability in the context of cancer, and how our current knowledge may be exploited for developing cancer therapies.

Mild replication stress causes chromosome mis-segregation via premature centriole disengagement.

Replication stress, a hallmark of cancerous and pre-cancerous lesions, is linked to structural chromosomal aberrations. Recent studies demonstrated that it could also lead to numerical chromosomal instability (CIN). The mechanism, however, remains elusive. Here, we show that inducing replication stress in non-cancerous cells stabilizes spindle microtubules and favours premature centriole disengagement, causing transient multipolar spindles that lead to lagging chromosomes and micronuclei. Premature centriole disengagement depends on the G2 activity of the Cdk, Plk1 and ATR kinases, implying a DNA-damage induced deregulation of the centrosome cycle. Premature centriole disengagement also occurs spontaneously in some CIN+ cancer cell lines and can be suppressed by attenuating replication stress. Finally, we show that replication stress potentiates the effect of the chemotherapeutic agent taxol, by increasing the incidence of multipolar cell divisions. We postulate that replication stress in cancer cells induces numerical CIN via transient multipolar spindles caused by premature centriole disengagement.

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

Mammalian 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.

ERCC1 and MUS81-EME1 promote sister chromatid separation by processing late replication intermediates at common fragile sites during mitosis.

Chromosomal instability (CIN) is a hallmark of tumour initiation and progression. Some genomic regions are particularly unstable under replication stress, notably common fragile sites (CFSs) whose rearrangements in tumour cells contribute to cancer development. Recent work has shown that the Fanconi anaemia (FANC) pathway plays a role in preventing defective chromosome segregation and CIN under conditions of replication stress. Strikingly, FANCD2 is recruited to regions hosting CFSs on metaphase chromosomes. To decipher the mechanisms protecting CFSs in G2/M, we searched for proteins that co-localize with FANCD2 on mitotic chromosomes, and identified XPF-ERCC1 and MUS81-EME1, two structure-specific endonucleases. We show that depletion of either ERCC1 or MUS81-EME1 affects accurate processing of replication intermediates or under-replicated DNA that persist at CFSs until mitosis. Depletion of these endonucleases also leads to an increase in the frequency of chromosome bridges during anaphase that, in turn, favours accumulation of DNA damage in the following G1 phase.

Replication dynamics: biases and robustness of DNA fiber analysis.

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.

AKT1/BRCA1 in the control of homologous recombination and genetic stability: the missing link between hereditary and sporadic breast cancers.

Endogenous replicative stress could be one trigger leading to tumor initiation: indeed, activation of the DNA damage response (DDR), considered the result of replicative stress, is observed in pre-cancerous cells; moreover, in hereditary breast cancers, almost all of the genes affected relate to the DDR. The most frequently mutated gene in hereditary breast cancers, BRCA1, is essential for homologous recombination (HR), a fundamental process for maintaining genome stability that permits the reactivation of blocked replication forks . Recent studies have established links between DDR and the oncogenic kinase AKT1, which is upregulated in about 50% of sporadic breast cancers. More specifically, the activation of AKT1 shows a deficient phenotype in BRCA1 and HR, revealing molecular similarities between hereditary and sporadic breast cancers. However, these results reveal a paradox regarding the physiological role of AKT1: in non-tumor cells, AKT1 promotes cellular proliferation, but consequently endangers genome integrity during replication if HR is inhibited. Since HR could itself lead to genetic instability, we propose that, under physiological conditions, moderate activation of AKT1 does not inhibit but prevents an excess of HR. The regulation of AKT1 would represent a fine transitory system for controlling HR and maintaining genomic integrity.