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Nucleic Acids Research Oct 2023Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source...
Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.
Topics: Humans; Cell Cycle Proteins; Chromatin; Congenital Microtia; Cyclin-Dependent Kinases; DNA Replication; Growth Disorders; Micrognathism; Minichromosome Maintenance Proteins; Patella; Replication Origin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37638758
DOI: 10.1093/nar/gkad694 -
Nature Communications Aug 2023The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of...
The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.
Topics: Animals; Adenoviridae; Cell Nucleus; Chromatin; Genomics; Mammals; NFI Transcription Factors; Nucleosomes; Humans
PubMed: 37604829
DOI: 10.1038/s41467-023-40846-1 -
The EMBO Journal Mar 2024Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and...
Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.
Topics: DNA; DNA Replication; DNA-Binding Proteins; MRE11 Homologue Protein; Rad51 Recombinase; Humans
PubMed: 38360996
DOI: 10.1038/s44318-024-00038-z -
Current Opinion in Microbiology Feb 2024All cells must ensure precise regulation of DNA replication initiation in coordination with growth rate and in response to nutrient availability. According to a... (Review)
Review
All cells must ensure precise regulation of DNA replication initiation in coordination with growth rate and in response to nutrient availability. According to a long-standing model, DNA replication initiation is tightly coupled to cell mass increase in bacteria. Despite controversies regarding this model, recent studies have provided additional support of this idea. The exact molecular mechanisms linking cell growth with DNA replication under different nutrient conditions remain elusive. However, recent studies in Caulobacter crescentus and Escherichia coli have provided insights into the regulation of DNA replication initiation in response to starvation. These mechanisms include the starvation-dependent regulation of DnaA abundance as well as mechanisms involving the small signaling molecule (p)ppGpp. In this review, we discuss these mechanisms in the context of previous findings. We highlight species-dependent similarities and differences and consider the precise growth conditions, in which the different mechanisms are active.
Topics: DNA-Binding Proteins; DNA, Bacterial; Bacterial Proteins; DNA Replication; Escherichia coli; Caulobacter crescentus
PubMed: 38035509
DOI: 10.1016/j.mib.2023.102403 -
Cell Communication and Signaling : CCS Jun 2023Cancer is a major cause of high morbidity and mortality worldwide. Several environmental, genetic and lifestyle factors are associated with the development of cancer in... (Review)
Review
Cancer is a major cause of high morbidity and mortality worldwide. Several environmental, genetic and lifestyle factors are associated with the development of cancer in humans and result in suboptimal treatment. The human microbiota has been implicated in the pathophysiological process of cancer and has been used as a diagnostic, prognostic and risk assessment tool in cancer management. Notably, both extratumoural and intratumoural microbiota are important components of the tumor microenvironment, subtly influencing tumorigenesis, progression, treatment and prognosis. The potential oncogenic mechanisms of action of the intratumoural microbiota include induction of DNA damage, influence on cell signaling pathways and impairment of immune responses. Some naturally occurring or genetically engineered microorganisms can specifically accumulate and replicate in tumors and then initiate various anti-tumor programs, ultimately promoting the therapeutic effect of tumor microbiota and reducing the toxic and side effects of conventional tumor treatments, which may be conducive to the pursuit of accurate cancer treatment. In this review, we summarise evidence revealing the impact of the intratumoural microbiota on cancer occurrence and progress and potential therapeutic and diagnostic applications, which may be a promising novel strategy to inhibit tumor development and enhance therapeutic efficacy. Video Abstract.
Topics: Humans; Carcinogenesis; Cell Transformation, Neoplastic; DNA Damage; Microbiota; Tumor Microenvironment
PubMed: 37381018
DOI: 10.1186/s12964-023-01134-z -
Nucleic Acids Research Oct 2023Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair....
Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.
Topics: DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Protein Binding; Replication Protein A; Saccharomyces cerevisiae; Bacteriophage T4
PubMed: 37739410
DOI: 10.1093/nar/gkad765 -
Genes & Development Oct 2023Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN...
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.
Topics: Humans; DNA Replication; DNA Helicases; Microsatellite Repeats; DNA Damage; Neoplasms; RecQ Helicases; Exodeoxyribonucleases; Werner Syndrome Helicase; Ataxia Telangiectasia Mutated Proteins
PubMed: 37932011
DOI: 10.1101/gad.351085.123 -
Science Advances Oct 2023Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects...
Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.
Topics: Humans; Cell Cycle Proteins; Cell Line, Tumor; Nuclear Proteins; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Transcription Factors; Transcription, Genetic; DNA Topoisomerases, Type I
PubMed: 37831776
DOI: 10.1126/sciadv.adg5109