Chiara Conti

Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription

Topoisomerase I (Top1) is a key enzyme in functioning at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing a conflict between transcription and DNA replication. Using DNA combing and ChIP (chromatin immunoprecipitation)-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase, and that breaks occur preferentially at gene-rich regions of the genome. Notably, these phenotypes were suppressed by preventing the formation of RNA–DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2), which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during the early stages of tumorigenesis.

The Intra-S-Phase Checkpoint Affects both DNA Replication Initiation and Elongation: Single-Cell and -DNA Fiber Analyses

ABSTRACT To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone γ-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.

Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks

Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)—which are potent transcription‐blocking lesions—also produce transcription‐dependent DNA double‐strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post‐mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, γ‐H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB–ATM–DDR pathway was suppressed by inhibiting transcription and γ‐H2AX signals were reduced by RNase H1 transfection, which removes transcription‐mediated R‐loops. Thus, we propose that Top1cc produce transcription arrests with R‐loop formation and generate DSBs that activate ATM in post‐mitotic cells.

Inhibition of Histone Deacetylase in Cancer Cells Slows Down Replication Forks, Activates Dormant Origins, and Induces DNA Damage

Protein acetylation is a reversible process regulated by histone deacetylases (HDAC) that is often altered in human cancers. Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and single-replicon analyses were conducted by genome-wide molecular combing after pulse labeling with two thymidine analogues. We found that pharmacologic concentrations of SAHA induce replication-mediated DNA damage with activation of histone gammaH2AX. Single DNA molecule analyses indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the beta-globin locus control region. Our findings demonstrate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells.

Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase.

Checkpoint kinase 1 (Chk1) regulates cell cycle checkpoints and DNA damage repair in response to genotoxic stress. Inhibition of Chk1 is an emerging strategy for potentiating the cytotoxicity of chemotherapeutic drugs. Here, we demonstrate that AZD7762, an ATP-competitive Chk1/2 inhibitor induces γ-H2AX in gemcitabine-treated cells by altering both dynamics and stability of replication forks, allowing the firing of suppressed replication origins as measured by DNA fiber combing and causing a dramatic increase in DNA breaks as measured by comet assay. Furthermore, we identify ATM and DNA-PK, rather than ATR, as the kinases mediating γ-H2AX induction, suggesting AZD7762 converts stalled forks into double strand breaks (DSBs). Consistent with DSB formation upon fork collapse, cells deficient in DSB repair by lacking BRCA2, XRCC3, or DNA-PK were selectively more sensitive to combined AZD7762 and gemcitabine. Checkpoint abrogation by AZD7762 also caused premature mitosis in gemcitabine-treated cells arrested in G1/early S-phase. Prevention of premature mitotic entry via Cdk1 siRNA knockdown suppressed apoptosis. These results demonstrate that chemosensitization of gemcitabine by Chk1 inhibition results from at least three cellular events namely activation of origin firing, destabilization of stalled replication forks, and entry of cells with damaged DNA into lethal mitosis. Additionally, the current study indicates that the combination of Chk1 inhibitor and gemcitabine may be particularly effective in targeting tumors with specific DNA repair defects.

The mammalian DNA replication elongation checkpoint: implication of Chk1 and relationship with origin firing as determined by single DNA molecule and single cell analyses.

The regulation of DNA replication initiation is well documented, for both unperturbed and damaged cells. The regulation of elongation, or fork velocity, however, has only recently been revealed with the advent of new techniques allowing us to view DNA replication at the single cell and single DNA molecule levels. Normally in S phase, the progression of replication forks and their stability are regulated by the ATR-Claspin-Chk1 pathway. We recently showed that replication fork velocity varies across the human genome in normal and cancer cells, but that the velocity of a given fork is positively correlated with the distance between origins on the same DNA fiber. Accordingly, in DNA replication-deficient Bloom’s syndrome cells, reduced fork velocity is associated with an increased density of replication origins. Replication elongation is also regulated in response to DNA damage. In human colon carcinoma cells treated with the topoisomerase I inhibitor camptothecin, DNA replication is inhibited both at the level of initiation and at the level of elongation through a Chk1-dependent checkpoint mechanism. Together, these new findings demonstrate that replication fork velocity (fork progression) is coordinated with inter-origin distance and that it can be actively slowed down by Chk1-dependent mechanisms in response to DNA damage. Thus, we propose that the intra-S phase checkpoint consist of at least three elements: (1) stabilization of damaged replication forks; (2) suppression of firing of late origins; and (3) arrests of normal ongoing forks to prevent further DNA lesions by replication of a damaged DNA template.

Upregulation of Error-Prone DNA Polymerases Beta and Kappa Slows Down Fork Progression Without Activating the Replication Checkpoint

There is rising evidence that cancer development is associated from its earliest stages with DNA replication stress, a major source of spontaneous genomic instability. However, the origin of these replication defects has remained unclear. We have investigated the consequences of upregulating error-prone DNA polymerases (pol) beta and kappa on chromosomal DNA replication. These enzymes are misregulated in different types of cancers and induce major chromosomal instabilities when overexpressed at low levels. Here, we have used DNA combing to show that a moderate overexpression of pol beta or pol kappa is sufficient to impede replication fork progression and to promote the activation of additional replication origins. Interestingly, alterations of the normal replication program induced by excess error-prone polymerases were not detected by the replication checkpoint. We therefore propose that upregulation of error-prone DNA polymerases induces a checkpoint-blind replication stress that contributes to genomic instability and to cancer development.

Methods to study replication fork collapse in budding yeast.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage. Hence, the comprehension of the mechanisms that preserve replication fork integrity or that promote fork collapse are extremely relevant for the understanding of the cellular processes controlling genome stability. Here we describe some experimental approaches that can be used to physically visualize the quality of replication forks in the yeast S. cerevisiae and to distinguish between stalled and collapsed forks.

Combing the genome for genomic instability

Genomic instability is one of the major features of cancer cells. The clinical phenotypes associated with several human diseases have been linked to recurrent DNA rearrangements and dysfunction of DNA replication processes that involve unstable genomic regions. Analysis of these rearrangements, which are frequently submicroscopic and can lead to loss or gain of dosage-sensitive genes or gene disruption, requires the development of sensitive, high-resolution techniques. This will lead to a better understanding of the mechanisms underlying genome instability and a greater awareness of the role of chromosomal rearrangements in disease. A new technology that involves molecular combing, a method that permits straightening and aligning molecules of genomic DNA, should make possible a detailed analysis of genomic events at the level of single DNA molecules. Such a single molecule approach could help to elucidate important properties that are masked in bulk studies.

Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon.

Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV‐18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.