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Nature Communications Nov 2022DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been...
DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.
Topics: Humans; DNA Repair; Genomic Instability; DNA Replication; S Phase; Cell Cycle
PubMed: 36400763
DOI: 10.1038/s41467-022-34379-2 -
Advances in Experimental Medicine and... 2017Eukaryotic genomes are replicated starting from multiple origins of replication. Their usage is tightly regulated, and not all the potential origins are activated during... (Review)
Review
Eukaryotic genomes are replicated starting from multiple origins of replication. Their usage is tightly regulated, and not all the potential origins are activated during a single cell cycle. In addition, the ones that are activated are activated in a sequential order. Why don't origins of replication normally all fire together? Is this important? And if so, why? Would any order of firing do, or does the specific sequence matter? How is this process regulated? These questions concern all eukaryotes but have proven extremely hard to address because replication timing is a process intricately connected with multiple aspects of nuclear function.
Topics: Animals; Cell Cycle Proteins; Cell Division; DNA Replication; DNA Replication Timing; Genome; Genomic Instability; Humans; Mammals; Telomere-Binding Proteins
PubMed: 29357062
DOI: 10.1007/978-981-10-6955-0_12 -
ACS Synthetic Biology Jun 2023Recent advances in synthetic biology have made it possible to reconstitute various cellular functions in a test tube. However, the integration of these functions...
Recent advances in synthetic biology have made it possible to reconstitute various cellular functions in a test tube. However, the integration of these functions remains a major challenge. This study aimed to identify a suitable condition to achieve all three reactions that constitute the central dogma: transcription, translation, and DNA replication. Specifically, we investigated the effect of the concentrations of 11 nonprotein factors required for transcription, translation, and DNA replication on each of these reactions. Our results indicate that certain factors have opposing effects on the three reactions. For example, while dNTP is necessary for DNA replication, it inhibited translation, and both rNTP and tRNA, which are essential for transcription and translation, inhibited DNA replication with several DNA polymerases. We also found that these opposing effects were partially alleviated by optimizing the magnesium concentration. Using this knowledge, we successfully demonstrated transcription/translation-coupled DNA replication with higher levels of transcription and translation while maintaining a certain level of DNA replication. These findings not only provide useful insights for the development of a complex artificial system with the central dogma but also raise the question of how natural cells overcome the incompatibility between the three reactions.
Topics: DNA Replication; DNA-Directed DNA Polymerase; Synthetic Biology
PubMed: 37271965
DOI: 10.1021/acssynbio.3c00130 -
Nature Communications Aug 2018Discriminating the contribution of unmodifiable random intrinsic DNA replication errors ('bad luck') to cancer development from those of other factors is critical for... (Review)
Review
Discriminating the contribution of unmodifiable random intrinsic DNA replication errors ('bad luck') to cancer development from those of other factors is critical for understanding cancer in humans and for directing public resources aimed at reducing the burden of cancer. Here, we review and highlight the evidence that demonstrates cancer causation is multifactorial, and provide several important examples where modification of risk factors has achieved cancer prevention. Furthermore, we stress the need and opportunities to advance understanding of cancer aetiology through integration of interaction effects between risk factors when estimating the contribution of individual and joint factors to cancer burden in a population. We posit that non-intrinsic factors drive most cancer risk, and stress the need for cancer prevention.
Topics: Animals; DNA Replication; Humans; Neoplasms; Risk Factors
PubMed: 30154431
DOI: 10.1038/s41467-018-05467-z -
Nature Structural & Molecular Biology Jan 2019Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly...
Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5' ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3' ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.
Topics: DNA Replication; Humans; Polyadenylation; RNA Polymerase II; Replication Origin; Transcription Initiation Site; Transcription, Genetic
PubMed: 30598550
DOI: 10.1038/s41594-018-0171-0 -
Open Biology Apr 2018Oncogene activation during tumour development leads to changes in the DNA replication programme that enhance DNA replication stress. Certain regions of the human genome,... (Review)
Review
Oncogene activation during tumour development leads to changes in the DNA replication programme that enhance DNA replication stress. Certain regions of the human genome, such as common fragile sites and telomeres, are particularly sensitive to DNA replication stress due to their inherently 'difficult-to-replicate' nature. Indeed, it appears that these regions sometimes fail to complete DNA replication within the period of interphase when cells are exposed to DNA replication stress. Under these conditions, cells use a salvage pathway, termed 'mitotic DNA repair synthesis (MiDAS)', to complete DNA synthesis in the early stages of mitosis. If MiDAS fails, the ensuing mitotic errors threaten genome integrity and cell viability. Recent studies have provided an insight into how MiDAS helps cells to counteract DNA replication stress. However, our understanding of the molecular mechanisms and regulation of MiDAS remain poorly defined. Here, we provide an overview of how DNA replication stress triggers MiDAS, with an emphasis on how common fragile sites and telomeres are maintained. Furthermore, we discuss how a better understanding of MiDAS might reveal novel strategies to target cancer cells that maintain viability in the face of chronic oncogene-induced DNA replication stress.
Topics: Chromosome Fragile Sites; DNA Repair; DNA Replication; Genomic Instability; Humans; Neoplasms; Telomere Homeostasis
PubMed: 29695617
DOI: 10.1098/rsob.180018 -
Environmental and Molecular Mutagenesis Aug 2020Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced... (Review)
Review
Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can "collapse" to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia-mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal "back-up" roles for Chk2 in S-phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents.
Topics: Animals; Carcinogenesis; Checkpoint Kinase 2; DNA Damage; DNA Replication; Environmental Exposure; Humans; Neoplasms
PubMed: 32578892
DOI: 10.1002/em.22397 -
Advances in Protein Chemistry and... 2019Genomically instable cancers are characterized by progressive loss and gain of chromosomal fragments, and the acquisition of complex genomic rearrangements. Such... (Review)
Review
Genomically instable cancers are characterized by progressive loss and gain of chromosomal fragments, and the acquisition of complex genomic rearrangements. Such cancers, including triple-negative breast cancers and high-grade serous ovarian cancers, typically show aggressive behavior and lack actionable driver oncogenes. Increasingly, oncogene-induced replication stress or defective replication fork maintenance is considered an important driver of genomic instability. Paradoxically, while replication stress causes chromosomal instability and thereby promotes cancer development, it intrinsically poses a threat to cellular viability. Apparently, tumor cells harboring high levels of replication stress have evolved ways to cope with replication stress. As a consequence, therapeutic targeting of such compensatory mechanisms is likely to preferentially target cancers with high levels of replication stress and may prove useful in potentiating chemotherapeutic approaches that exert their effects by interfering with DNA replication. Here, we discuss how replication stress drives chromosomal instability, and the cell cycle-regulated mechanisms that cancer cells employ to deal with replication stress. Importantly, we discuss how mechanisms involving DNA structure-specific resolvases, cell cycle checkpoint kinases and mitotic processing of replication intermediates offer possibilities in developing treatments for difficult-to-treat genomically instable cancers.
Topics: Antineoplastic Agents; DNA Replication; Genomic Instability; Humans; Neoplasms; Stress, Physiological
PubMed: 30798931
DOI: 10.1016/bs.apcsb.2018.10.006 -
Proceedings of the National Academy of... May 2020
Topics: DNA; DNA Replication; DNA-Directed DNA Polymerase; Mutation; Saccharomyces cerevisiae
PubMed: 32350136
DOI: 10.1073/pnas.2005160117 -
Chemical Reviews Dec 2023The paradigm of cellular systems as deterministic machines has long guided our understanding of biology. Advancements in technology and methodology, however, have... (Review)
Review
The paradigm of cellular systems as deterministic machines has long guided our understanding of biology. Advancements in technology and methodology, however, have revealed a world of stochasticity, challenging the notion of determinism. Here, we explore the stochastic behavior of multi-protein complexes, using the DNA replication system (replisome) as a prime example. The faithful and timely copying of DNA depends on the simultaneous action of a large set of enzymes and scaffolding factors. This fundamental cellular process is underpinned by dynamic protein-nucleic acid assemblies that must transition between distinct conformations and compositional states. Traditionally viewed as a well-orchestrated molecular machine, recent experimental evidence has unveiled significant variability and heterogeneity in the replication process. In this review, we discuss recent advances in single-molecule approaches and single-particle cryo-EM, which have provided insights into the dynamic processes of DNA replication. We comment on the new challenges faced by structural biologists and biophysicists as they attempt to describe the dynamic cascade of events leading to replisome assembly, activation, and progression. The fundamental principles uncovered and yet to be discovered through the study of DNA replication will inform on similar operating principles for other multi-protein complexes.
Topics: DNA Replication; DNA; Molecular Conformation
PubMed: 37971892
DOI: 10.1021/acs.chemrev.3c00436