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Nature Cell Biology Aug 2022Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but...
Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.
Topics: Cell Cycle; Cell Division; DNA Replication; Mechanistic Target of Rapamycin Complex 1; Nucleotides; S Phase
PubMed: 35927450
DOI: 10.1038/s41556-022-00965-1 -
Molecular Cell Sep 2022Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements....
Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.
Topics: Chromosomes; DNA Replication; Humans; Rad51 Recombinase; S Phase; Transcription, Genetic
PubMed: 36002000
DOI: 10.1016/j.molcel.2022.07.010 -
Nature Communications Jun 2022R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to...
R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. ZFP281 depletion reduces PCNA levels on chromatin and impairs DNA replication. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.
Topics: Animals; Chromatin; DNA Replication; Embryonic Stem Cells; Genomic Instability; Mammals; R-Loop Structures; Transcription Factors
PubMed: 35715464
DOI: 10.1038/s41467-022-31211-9 -
Cells Feb 2022All living organisms on Earth are made up of cells, which are the functional unit of life. Eukaryotic organisms can consist of a single cell (unicellular) or a group of... (Review)
Review
All living organisms on Earth are made up of cells, which are the functional unit of life. Eukaryotic organisms can consist of a single cell (unicellular) or a group of either identical or different cells (multicellular). Biologists have always been fascinated by how a single cell, such as an egg, can give rise to an entire organism, such as the human body, composed of billions of cells, including hundreds of different cell types. This is made possible by cell division, whereby a single cell divides to form two cells. During a symmetric cell division, a mother cell produces two daughter cells, while an asymmetric cell division results in a mother and a daughter cell that have different fates (different morphologies, cellular compositions, replicative potentials, and/or capacities to differentiate). In biology, the cell cycle refers to the sequence of events that a cell must go through in order to divide. These events, which always occur in the same order, define the different stages of the cell cycle: G1, S, G2, and M. What is fascinating about the cell cycle is its universality, and the main reason for this is that the genetic information of the cell is encoded by exactly the same molecular entity with exactly the same structure: the DNA double helix. Since both daughter cells always inherit their genetic information from their parent cell, the underlying fundamentals of the cell cycle-DNA replication and chromosome segregation-are shared by all organisms. This review goes back in time to provide a historical summary of the main discoveries that led to the current understanding of how cells divide and how cell division is regulated to remain highly reproducible.
Topics: Cell Cycle; Cell Cycle Checkpoints; Cell Division; DNA Replication; Humans
PubMed: 35203358
DOI: 10.3390/cells11040704 -
Nature May 2022The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division. According to current models of the cell cycle, the kinase...
The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division. According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication.
Topics: Animals; Cell Cycle Proteins; DNA Replication; G1 Phase; Mice; Protein Serine-Threonine Kinases; Proteolysis; S Phase
PubMed: 35508654
DOI: 10.1038/s41586-022-04698-x -
Nature Apr 2022Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of...
Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.
Topics: Chromosomal Instability; DNA Damage; DNA Replication; Gene Duplication; Humans; Karyotype; Mitosis; S Phase; Tetraploidy
PubMed: 35355016
DOI: 10.1038/s41586-022-04578-4 -
Molecular Cell Dec 2022POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR...
POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.
Topics: Humans; DNA Breaks, Double-Stranded; DNA Replication; Genomic Instability; DNA, Single-Stranded; Synthetic Lethal Mutations; DNA End-Joining Repair; Neoplasms
PubMed: 36455556
DOI: 10.1016/j.molcel.2022.11.008 -
Cold Spring Harbor Perspectives in... Jul 2021Immediately following the discovery of the structure of DNA and the semi-conservative replication of the parental DNA sequence into two new DNA strands, it became... (Review)
Review
Immediately following the discovery of the structure of DNA and the semi-conservative replication of the parental DNA sequence into two new DNA strands, it became apparent that DNA replication is organized in a temporal and spatial fashion during the S phase of the cell cycle, correlated with the large-scale organization of chromatin in the nucleus. After many decades of limited progress, technological advances in genomics, genome engineering, and imaging have finally positioned the field to tackle mechanisms underpinning the temporal and spatial regulation of DNA replication and the causal relationships between DNA replication and other features of large-scale chromosome structure and function. In this review, we discuss these major recent discoveries as well as expectations for the coming decade.
Topics: Animals; DNA Packaging; DNA Replication Timing; Genome; Humans; Mammals
PubMed: 33558366
DOI: 10.1101/cshperspect.a040162 -
Nature Apr 2024An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination...
An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.
Topics: Humans; DNA Breaks, Double-Stranded; DNA Replication; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Recombinational DNA Repair; S Phase; Transcription, Genetic; Neoplasms; Poly (ADP-Ribose) Polymerase-1
PubMed: 38509368
DOI: 10.1038/s41586-024-07217-2 -
Molecular Cell Jan 2024Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise in each cell each day, many of which can directly or indirectly... (Review)
Review
Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise in each cell each day, many of which can directly or indirectly impede transcription. Conversely, the process of gene expression is itself a source of endogenous DNA lesions as a result of the susceptibility of single-stranded DNA to damage, conflicts with the DNA replication machinery, and engagement by cells of topoisomerases and base excision repair enzymes to regulate the initiation and progression of gene transcription. Although such processes are tightly regulated and normally accurate, on occasion, they can become abortive and leave behind DNA breaks that can drive genome rearrangements, instability, or cell death.
Topics: Humans; DNA Damage; DNA Replication; DNA Repair; DNA; Genome; Genomic Instability; Transcription, Genetic
PubMed: 38103560
DOI: 10.1016/j.molcel.2023.11.014