Initiation Of DNA Replication Research Articles

The initiation of mitochondrial DNA replication.

Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.

Pioneer factors for DNA replication initiation in metazoans.

Precise DNA replication is fundamental for genetic inheritance. In eukaryotes, replication initiates at multiple origins that are first "licensed" and subsequently "fired" to activate DNA synthesis. Despite the success in identifying origins with specific DNA motifs in Saccharomyces cerevisiae, no consensus sequence or sequences with a predictive value of replication origins have been recognized in metazoan genomes. Rather, epigenetic rules and chromatin structures are believed to play important roles in governing the selection and activation of replication origins. We propose that replication initiation is facilitated by a group of sequence-specific "replication pioneer factors," which function to increase chromatin accessibility and foster a chromatin environment that is conducive to the loading of the prereplication complex. Dysregulation of the function of these factors may lead to gene duplication, genomic instability, and ultimately the occurrence of pathological conditions such as cancer.

Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks.

CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4 or DRF1 for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated isogenic cell lines deficient in either DBF4 or DRF1: these cells are viable but present signs of genomic instability, indicating that both can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, partial deficiency in MCM helicase phosphorylation, and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not on DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, mediating most of its functions in unperturbed DNA replication and upon replication interference.

Functional studies in yeast confirm the pathogenicity of a new GINS3 Meier-Gorlin syndrome variant.

Meier-Gorlin syndrome (MGORS) is an autosomal recessive disorder characterized by short stature, microtia, and patellar hypoplasia, and is caused by pathogenic variants of cellular factors involved in the initiation of DNA replication. We previously reported that biallelic variants in GINS3 leading to amino acid changes at position 24 (p.Asp24) cause MGORS. Here, we describe the phenotype of a new individual homozygous for the Asp24Asn variant. We also report the clinical characteristics of an individual harboring a novel homozygous GINS3 variant (Ile25Phe) and features suggestive of MGORS. Modification of the corresponding residue in yeast Psf3 (Val9Phe) compromised S phase progression compared to a humanized Psf3 Val9Ile variant. Expression of Psf3 Val9Phe in yeast also caused sensitivity to elevated temperature and the replicative stress-inducing drug hydroxyurea, confirming partial loss of function of this variant in vivo and allowing us to upgrade the classification of this variant. Taken together, these data validate the critical importance of the GINS DNA replication complex in the molecular etiology of MGORS.

Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.

Proteomics Analysis of the Polyomavirus DNA Replication Initiation Complex Reveals Novel Functional Phosphorylated Residues and Associated Proteins.

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

Targeting synthesis of the Chromosome Replication Initiator Protein DnaA by antisense PNA-peptide conjugates in Escherichia coli

Initiation of chromosome replication is an essential stage of the bacterial cell cycle that is controlled by the DnaA protein. With the aim of developing novel antimicrobials, we have targeted the initiation of DNA replication, using antisense peptide nucleic acids (PNAs), directed against DnaA translation. A series of anti-DnaA PNA conjugated to lysine-rich bacterial penetrating peptides (PNA-BPPs) were designed to block DnaA translation. These anti-DnaA PNA-BPPs inhibited growth of wild-type Escherichia coli cells at low micromolar concentrations, and cells exposed to anti-DnaA PNA-BPPs exhibited characteristic hallmarks of chromosome replication inhibition. These results present one of very few compounds successfully targeting initiation of chromosome replication, an essential step in the bacterial cell cycle.

Three factors ParA, TipN, and DnaA-mediated chromosome replication initiation are contributors of centromere segregation in Caulobacter crescentus.

The ability of bacteria to maintain chromosomal integrity throughout their life cycle is crucial for survival. In Caulobacter crescentus, the polar factor TipN has been proposed to be involved with the partitioning system ParABS. Cells with tipN knocked out display subtle segregation defects of the centromere-like region parS. We hypothesized that TipN's role with parS segregation is obscured by other forces that are ParABS-independent. To test our hypothesis, we removed one of those forces - chromosome replication - and analyzed the role of TipN with ParA. We first confirm that ParA retains its ability to transport the centromeric region parS from the stalked pole to the opposite pole in the absence of chromosome replication. Our data revealed that in the absence of chromosome replication, TipN becomes essential for ParA's ability to transport parS. Furthermore, we identify a potential connection between the replication initiator DnaA and TipN. Although TipN is not essential for viability, tipN knockout cells lose viability when the regulation of DnaA levels is altered. Our data suggest that the DnaA-dependent susceptibility of tipN knockout cells is connected to parS segregation. Collectively, this work provides insights into the complex regulation involved in the coordination of chromosome replication and segregation in bacteria.

RECQL4 is not critical for firing of human DNA replication origins

Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.

A Decade of Discovery-Eukaryotic Replisome Disassembly at Replication Termination.

The eukaryotic replicative helicase (CMG complex) is assembled during DNA replication initiation in a highly regulated manner, which is described in depth by other manuscripts in this Issue. During DNA replication, the replicative helicase moves through the chromatin, unwinding DNA and facilitating nascent DNA synthesis by polymerases. Once the duplication of a replicon is complete, the CMG helicase and the remaining components of the replisome need to be removed from the chromatin. Research carried out over the last ten years has produced a breakthrough in our understanding, revealing that replication termination, and more specifically replisome disassembly, is indeed a highly regulated process. This review brings together our current understanding of these processes and highlights elements of the mechanism that are conserved or have undergone divergence throughout evolution. Finally, we discuss events beyond the classic termination of DNA replication in S-phase and go over the known mechanisms of replicative helicase removal from chromatin in these particular situations.

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest.

Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies.

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy.

Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein-DNA complex remains active in the solid-state NMR rotor and that time-resolved 31P-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved 1H-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.

Unwinding Helicase MCM Functionality for Diagnosis and Therapeutics of Replication Abnormalities Associated with Cancer: A Review.

The minichromosome maintenance (MCM) protein is a component of an active helicase that is essential for the initiation of DNA replication. Dysregulation of MCM functions contribute to abnormal cell proliferation and genomic instability. The interactions of MCM with cellular factors, including Cdc45 and GINS, determine the formation of active helicase and functioning of helicase. The functioning of MCM determines the fate of DNA replication and, thus, genomic integrity. This complex is upregulated in precancerous cells and can act as an important tool for diagnostic applications. The MCM protein complex can be an important broad-spectrum therapeutic target in various cancers. Investigations have supported the potential and applications of MCM in cancer diagnosis and its therapeutics. In this article, we discuss the physiological roles of MCM and its associated factors in DNA replication and cancer pathogenesis.

Abstract 5714: Synergistic effect of the CDC7 inhibitor, monzosertib (AS-0141) with current therapies in AML models

Abstract Introduction: Cell division cycle 7 (CDC7) is a highly conserved serine-threonine kinase that plays an important role in the initiation of DNA replication and cell cycle progression. Aberrant expression of CDC7 have been reported to cause uncontrolled proliferation of many cancer types, suggesting that CDC7 inhibitors may provide a great potential for the development of novel therapy for cancers. Monzosertib (AS-0141) is a potent, selective, orally bioavailable small molecule inhibitor of CDC7, and is currently being evaluated in Phase I study for the treatment of solid tumors. In a preclinical cancer cell panel study, monzosertib showed strong antiproliferative activities against a variety of cancer types, and acute myeloid lymphoma (AML) cell lines were found to be the most sensitive to monzosertib. As a single agent, treatment with monzosertib accumulates DNA damage in cancer cells and induces cell death. In this study, we aimed to investigate the antitumor effects of monzosertib alone and in combination with other anticancer drugs in AML models. Method: Antiproliferative activity of monzosertib was examined against a panel of 35 human cancer cell lines of various cancer types. DNA metyltransferase (DNMT) inhibitors (azacitidine or decitabine) and BCL2 inhibitor (venetoclax) were evaluated for their synergistic/antagonistic effects in combination with monzosertib against human AML cell lines (THP-1, HL-60, MV4-11, MOLM-14, TF-1, U-937 and NOMO-1). The combination index (CI) was calculated using the Chou-Talalay method. Flow cytometry assay was used to analyze apoptosis. To evaluate the in vivo efficacy, tumor-bearing mice were treated with monzosertib alone or in combination with venetoclax. Results: The combination of monzosertib with azacitidine, decitabine, or venetoclax resulted in a significant synergistic antiproliferative effects against AML cell lines in vitro. The flow cytometry assay indicated that azacitidine combination induced apoptosis and increased cell death in THP-1 cells. In vivo, oral administration of monzosertib demonstrated robust in vivo antitumor efficacy in a MV4-11 tumor bearing xenograft mouse model, both as a single agent and in combination with venetoclax. Conclusions: Monzosertib, a selective CDC7 inhibitor, demonstrated strong antiproliferative activity against human AML cell lines, both as a single agent and in combination with standard therapies. Monzosertib exerts synergistic antitumor effect with venetoclax in a human AML xenograft mouse model. These results suggest that monzosertib has a potential to enhance the antitumor efficacy of standard of care agents for AML patients. Citation Format: Hiroko Endo, Hatsuo Furuichi, Akinori Arimura, Yu Nishioka, Masaaki Sawa. Synergistic effect of the CDC7 inhibitor, monzosertib (AS-0141) with current therapies in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5714.

A commentary of “Initial DNA melting in human DNA replication initiation”: Top 10 Scientific Advances of 2023, China

A commentary of “Initial DNA melting in human DNA replication initiation”: Top 10 Scientific Advances of 2023, China

Abstract 6988: TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer

Abstract Background: We identify TP53 regulating kinase (TP53RK), which showed the highest cell growth inhibition efficiency in six colon cancer cell lines. TP53RK is overexpressed in various cancer types, including multiple myeloma and skin cancer, but the mechanism for its tumorigenesis in colon cancer is unknown. Therefore, this study aims to reveal the tumorigenesis mechanism of TP53RK, which is overexpressed in Colorectal Cancer (CRC), through global and phospho-proteomics. Method: A total 5 CRC cell lines (HT29, SW480, HCT116, H508 and CaCO2), colon normal fibroblast CRL1459, HCT116 p53 null cell and patient-derived normal organoid were used. Immunoblotting, MTT assay, Colony formation assay, Annexin-V assay and cell cycle analysis were performed for evaluation of TP53RK loss-of-function. With kinase-substrate enrichment analysis with phospho-proteome analysis, MCM2 was found to be a substrate for the kinase regulated by TP53RK. Results: High-throughput genetic screening approach have been widely applied to the study the gene function and molecular mechanism associated with tumorigenesis. By using genome-wide CRISPR/Cas9 library screening, we identify TP53RK, the first committed negative-selected gene, as a tyrosine kinase in the colorectal cancer(CRC) specific biomarker. Through proteome analysis, we found that depletion of TP53RK can hypo-phosphorylate the DNA helicase complex, MCM2 (minichromosome maintenance protein2). According to previous studies, the phosphorylation of MCM2 is regulated by CDC7 (cell division cycle7), and the abnormal states of CDC7 and MCM2 cause DNA replication disorder. Our results show that TP53RK expression is upregulated in CRC and its depletion results in decreased CDC7 expression and hypo-phosphorylation of the MCM complex. Decreased CDC7 expression due to its instability after TP53RK depletion can lead to DNA replication errors. DNA replication errors can lead cancer cells to apoptosis, suggesting that they may be a strategy for cancer treatment. In summary, we provide TP53RK as a novel prognostic and therapeutic target in CRC. Conclusion: To recapitulate briefly, our findings suggest that the interaction of TP53RK with CDC7 can regulate CDC7 protein stability and stabilized CDC7 can phosphorylate MCM2 to initiate DNA replication. In other words, because overexpression of TP53RK acts as an oncogene that promotes tumor progression in CRC, depletion of TP53RK can degrade CDC7 and induce colon cancer cells to death or regression. Citation Format: Younghee Choi, Ji-won Park, Jun-kyu Kang, Eun-ju Kim, Sang-Hyun Song, Eugene C. Yi, Tae-You Kim. TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6988.

A second hotspot for pathogenic exon-skipping variants in CDC45.

Biallelic pathogenic variants in CDC45 are associated with Meier-Gorlin syndrome with craniosynostosis (MGORS type 7), which also includes short stature and absent/hypoplastic patellae. Identified variants act through a hypomorphic loss of function mechanism, to reduce CDC45 activity and impact DNA replication initiation. In addition to missense and premature termination variants, several pathogenic synonymous variants have been identified, most of which cause increased exon skipping of exon 4, which encodes an essential part of the RecJ-orthologue's DHH domain. Here we have identified a second cohort of families segregating CDC45 variants, where patients have craniosynostosis and a reduction in height, alongside common facial dysmorphisms, including thin eyebrows, consistent with MGORS7. Skipping of exon 15 is a consequence of two different variants, including a shared synonymous variant that is enriched in individuals of East Asian ancestry, while other variants in trans are predicted to alter key intramolecular interactions in α/β domain II, or cause retention of an intron within the 3'UTR. Our cohort and functional data confirm exon skipping is a relatively common pathogenic mechanism in CDC45, and highlights the need for alternative splicing events, such as exon skipping, to be especially considered for variants initially predicted to be less likely to cause the phenotype, particularly synonymous variants.

Lily Polysaccharide Nano‐Selenium Promotes A549 Cells Apoptosis through Cell Cycle Inhibition

AbstractThis study uses Lilium davidii var. unicolor (Lanzhou lily) polysaccharides extracted from bulbs to prepare nano‐selenium (nano‐Se) polysaccharide (LPS). Here, the study explores the molecular structure characterization, inhibition effects, and mechanisms of action of prepared LPS on A549 lung cancer cells. The LPS structure is characterized using Fourier‐transform infrared spectroscopy (FTIR), UV spectroscopy (UV–vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), X‐ray photoelectron spectroscopy (XPS), etc. Results show that selenized polysaccharides bonded through electrostatic adsorption, with an average LPS particle size of 243.75 nm and zeta potential of −38.3 mV, which can be stably preserved at 4 °C for more than 3 months. The in vitro antitumor activity experiment shows that the prepared LPS inhibited A549 cell proliferation while affecting cell migration. Cell cycle analysis shows that apoptosis is promoted by blocking the G1 phase, while during the late apoptosis stage cells accounted for 60%. RNA‐Seq data analysis shows that the main mechanisms of action on A549 cells are to inhibit DNA replication initiation genes, block the cell cycle, and regulate the expression of apoptosis‐related genes and proteins in promoting cell apoptosis.

Abstract 1259 Biochemical Investigation and Evolutionary Comparison of the Mechanism of the Initiation of DNA Replication in Eukaryotic Cells

Abstract 1259 Biochemical Investigation and Evolutionary Comparison of the Mechanism of the Initiation of DNA Replication in Eukaryotic Cells