-
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 -
International Journal of Molecular... May 2023Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that... (Review)
Review
Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.
Topics: Bacterial Proteins; DNA Replication; DNA-Binding Proteins; Bacteria; Protein Binding
PubMed: 37240152
DOI: 10.3390/ijms24108802 -
Trends in Genetics : TIG Nov 2020Genomic DNA is replicated every cell cycle by the programmed activation of replication origins at specific times and chromosomal locations. The factors that define the... (Review)
Review
Genomic DNA is replicated every cell cycle by the programmed activation of replication origins at specific times and chromosomal locations. The factors that define the locations of replication origins and their typical activation times in eukaryotic cells are poorly understood. Previous studies highlighted the role of activating factors and epigenetic modifications in regulating replication initiation. Here, we review the role that repressive pathways - and their alleviation - play in establishing the genomic landscape of replication initiation. Several factors mediate this repression, in particular, factors associated with inactive chromatin. Repression can support organized, yet stochastic, replication initiation, and its absence could explain instances of rapid and random replication or re-replication.
Topics: Animals; Cell Cycle; Chromatin; DNA Replication; Epigenesis, Genetic; Eukaryotic Cells; Humans; Origin Recognition Complex; Replication Origin
PubMed: 32739030
DOI: 10.1016/j.tig.2020.06.020 -
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 -
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 -
Nature Communications Jun 2021Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We...
Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.
Topics: Cell Cycle Proteins; Cell Line, Tumor; Cyclopentanes; DNA Replication; Genome, Human; Humans; Mitosis; Models, Biological; Pyrimidines; Replication Origin
PubMed: 34103496
DOI: 10.1038/s41467-021-23835-0 -
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 -
Nature Structural & Molecular Biology Mar 2023Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork...
Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork.
Topics: Humans; RNA; DNA Replication; DNA; DNA-Binding Proteins; Chromosomes; Genomic Instability
PubMed: 36864174
DOI: 10.1038/s41594-023-00928-6 -
International Journal of Molecular... May 2021In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for... (Review)
Review
In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4. The proximity of PCNA-bound Cdt1 to CRL4 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; DNA Replication; Genomic Instability; Humans
PubMed: 34068957
DOI: 10.3390/ijms22105195