Re-replication Research Articles

WRAD core perturbation impairs DNA replication fidelity promoting immunoediting in pancreatic cancer.

It is unclear how cells counteract the potentially harmful effects of uncoordinated DNA replication in the context of oncogenic stress. Here, we identify the WRAD (WDR5/RBBP5/ASH2L/DPY30) core as a modulator of DNA replication in pancreatic ductal adenocarcinoma (PDAC) models. Molecular analyses demonstrated that the WRAD core interacts with the replisome complex, with disruption of DPY30 resulting in DNA re-replication, DNA damage, and chromosomal instability (CIN) without affecting cancer cell proliferation. Consequently, in immunocompetent models, DPY30 loss induced T cell infiltration and immune-mediated clearance of highly proliferating cancer cells with complex karyotypes, thus improving anti-tumor efficacy upon anti-PD-1 treatment. In PDAC patients, DPY30 expression was associated with high tumor grade, worse prognosis, and limited response to immune checkpoint blockade. Together, our findings indicate that the WRAD core sustains genome stability and suggest that low intratumor DPY30 levels may identify PDAC patients who will benefit from immune checkpoint inhibitors.

Abstract 4694: In charge: Targeting neddylation of UBE2M for anticancer therapy

Abstract Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules orchestrates a vast number of processes in the cell. Consequently, post-translational modification pathways are considered major targets for therapeutic intervention of many diseases, including cancers. Neddylation is a post-translational mechanism like ubiquitination, but instead of ubiquitin, NEDD8 is conjugated to protein targets via an E1-E2-E3 multi-enzymatic cascade. Neddylation is upregulated in multiple cancer types and the development of therapeutics targeting neddylation pathways are gaining increasing popularity. UBE2M, one of the two E2 neddylation conjugation enzymes, takes part in two types of reactions: transthiolation-transfer of NEDD8 from a thioester to a thiol group and aminolysis-transfer of NEDD8 from a thioester to an amino group. These activities require both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of NEDD8. Here, we describe findings in the quest to understand the UBE2M interaction with NEDD8 and effects on cancer cells by modifying UBE2M at the N-terminal docking peptide or at the catalytic site. Cancer cells were transfected with ΔN or catalytic site UBE2M mutant constructs and analyzed by immunoprecipitation for charging of NEDD8 onto UBE2M. The catalytic site mutants are deficient in charging (C111A) or discharging (C111S) of UBE2M by NEDD8, which leads to a decrease in neddylation of downstream substrates, such as CULLIN family members. Elimination of the N-terminal docking peptide by introduction of catalytically competent ΔN UBE2M mutant constructs resulted in reduced reaction kinetics. Structural modeling suggests that the loss of the N-terminal docking region likely weakens protein-protein interactions between UBE2M and the UBA3-APPBP1 E1 complex. Interestingly, ΔN UBE2M mutants were observed to be slightly charged with NEDD8, but appeared to have a defect in NEDD8 discharge, resulting in reduced neddylation of substrates. Moreover, further phenotypic evaluation of UBE2M mutant transfected cancer cells revealed a divergence in phenotypic effect: while both mutant types led to a deficiency in NEDD8 charge or discharge from UBE2M, only the manipulation of the active site of UBE2M led to a DNA re-replication phenotype with subsequent cell death. These observations reveal the complexity of the neddylation cascade and raise the question if slowing down discharge of NEDD8 from UBE2M is a more viable therapeutic option than slowing down the charging of UBE2M. Citation Format: Alex G. Batrouni, Jacob P. Matson, Andressa L. Mota, Archna Ravi, Marcus O'Hara, Micholas D. Smith, Jeremy C. Smith, Vivek K. Vishnudas, Anjan Thakurta, Stephane Gesta, Maria D. Nastke. In charge: Targeting neddylation of UBE2M for anticancer therapy [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 4694.

Abstract A011: DPY30 regulates immunoediting by suppressing uncoordinated DNA replication in pancreatic cancer

Abstract DNA replication is a tightly regulated, multi-step process orchestrated by licensing and replication factors that ensure the faithful duplication of the entire genome exclusively during S phase of the cell cycle. Although the mechanisms preventing uncoordinated DNA replication are well characterized, it remains unclear how cells counteract its potentially harmful effects when it does occur, especially in a context of oncogenic stress. Here, we identify DPY30, an understudied component of all COMPASS-like complexes, as a modulator of DNA replication. We found upregulated DPY30 expression in pancreatic ductal adenocarcinoma (PDAC) as well as a correlation between increasing DPY30 expression level and higher tumor grade and poor clinical outcomes. To investigate the role of DPY30 in DNA replication, we generated and characterized human- and mouse-derived PDAC cells silenced or knockout for DPY30 expression in vivo, within a context of increasing immune pressure. ATAC-Seq and ChIP-Seq were used to inform on the epigenetic landscape of PDAC cells silenced for DPY30 or WDR5 expression. Additionally, we conducted unbiased proteomics and transcriptomics analyses to uncover the underlying molecular mechanisms. The canonical function of COMPASS complexes is to regulate histone 3 lysine 4 (H3K4) methylation. Unexpectedly, DPY30 loss in PDAC cells did not affect H3K4 methylation patterns compared to WDR5 loss, the core member of the COMPASS-like complex. Transcriptomic data unveil a dysregulation of genes belonging to DNA replication and repair pathways in DPY30-silenced PDAC cells. This prompted unbiased proteomics analysis, which showed that DPY30 interacts with members of the DNA helicase, possibly stabilizing the DNA replication fork progression. We found that DPY30 overexpression, by preventing Geminin ubiquitylation, strongly stabilizes its expression, which in turn suppresses Cdt1 activity in late S-phase, buffering the risk of uncoordinated DNA replication in G2/M phase. Consistently, DPY30 loss resulted in DNA re-replication and chromosomal instability, as manifested by DNA fiber and metaphasic spread. In vivo, whole-exome sequencing analysis of DPY30-knockout tumors revealed that DPY30 loss markedly increased mutational burden in immunodeficient mice. In syngeneic models, DPY30 loss strongly inhibited tumor growth, prolonged survival, and increased T cell infiltration of tumors. Moreover, the immune system selectively cleared tumor cells with complex karyotypes, and we observed improved anti-tumor efficacy with DPY30 knockdown and anti-PD-1 treatment compared to either condition alone. In summary, our study has unveiled a previously unrecognized role for DPY30 in stabilizing the replisome machinery by preventing excessive DNA replication. In the context of PDAC, DPY30 sustains genome stability, providing a strong rationale for exploring DPY30 or its effector proteins as potential targets and suggesting that patients whose tumors carry low DPY30 expression might be successfully treated with immune checkpoint inhibitors. Citation Format: Francesca Citron, Luigi Perelli, Chiara Balestrieri, Luca Cecchetto, Yanshuo Chu, I-Lin Ho, Li Zhang, Er-Yen Yen, Luis Castillo Montanez, Rutvi Shah, Sergio Attanasio, Sanjana Srinivasan, Charles Dyke, Ko-Chien Chen, Shan Jiang, Jing Pan, Sisi Gao, Huamin Wang, Wantong Yao, Linghua Wang, Mauro Di Pilato, Giannicola Genovese, Andrea Viale, Giulio Draetta. DPY30 regulates immunoediting by suppressing uncoordinated DNA replication in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A011.

Abstract 303: DPY30 loss leads to DNA re-replication and immunoediting in pancreatic ductal adenocarcinoma

Abstract Introduction: DNA replication is a tightly regulated, multi-step process involving licensing and replication factors that faithfully orchestrate a single duplication of the entire genome exclusively during S phase of the cell cycle. Although the mechanisms that prevent uncoordinated DNA replication are well characterized, it remains unclear how cells counteract its potentially harmful effects when it does occur, such as in cancer cells driven by aberrant oncogenic signaling and loss of cell cycle checkpoints. Here, we identify DPY30, an understudied component of all COMPASS complexes, as a modulator of DNA replication. We found upregulated DPY30 expression in pancreatic ductal adenocarcinoma (PDAC) as well as a correlation between increasing DPY30 expression level and higher tumor grade and poor clinical outcomes. Experimental procedure: Human- and mouse-derived PDAC cells silenced or knockout for DPY30 expression were generated and characterized for their in vivo growth in recipient mice with increasing level of immune pressure. Chromatin immunoprecipitation followed by massive parallel sequencing was used to measure the epigenetic landscape in DPY30 or WDR5 knockdown cells. Unbiased proteomics and transcriptomics analyses were performed to elucidate molecular mechanisms. Results: The canonical function of COMPASS complexes is to regulate histone methylation. Unexpectedly, DPY30 loss did not affect histone methylation patterns compared to WDR5 loss, the core member of the COMPASS-like complex. This prompted unbiased proteomics analysis, which showed that DPY30 interacts with members of the DNA helicase, possibly stabilizing the DNA replication fork progression. We found that DPY30 overexpression, by preventing Geminin ubiquitylation, strongly stabilizes its expression, which in turn suppresses Cdt1 activity in late S-phase, buffering the risk of uncoordinated DNA replication in G2/M phase. Consistently, DPY30 loss resulted in DNA re-replication and chromosomal instability, as manifested by DNA fiber and metaphasic spread. In vivo, whole-exome sequencing analysis of DPY30-knockout tumors revealed that DPY30 loss markedly increased mutational burden in immunodeficient mice. In syngeneic models, DPY30 loss strongly inhibited tumor growth, prolonged survival, and increased T cell infiltration of tumors. Moreover, the immune system selectively cleared tumor cells with complex karyotypes, and we observed improved anti-tumor efficacy with DPY30 knockdown and anti-PD-1 treatment compared to either condition alone. Conclusions: In summary, we have identified a previously undescribed function for DPY30 to stabilize the replisome machinery and prevent excessive DNA replication. Our findings indicate that, in PDAC, DPY30 promotes genome stability, thus providing a rationale for targeting DPY30 or its effector proteins in combination with immune-checkpoint inhibitors. Citation Format: Francesca Citron, Luigi Perelli, Yanshuo Chu, Sanjana Srinivasan, I-Lin Ho, Er-Yen Yen, Rutvi Shah, Sergio Attanasio, Luis Castillo Montanez, Mauro Di Pilato, Shan Jiang, Wantong Yao, Sisi Gao, Angela Deem, Giannicola Genovese, Linghua Wang, Virginia Giuliani, Andrea Viale, Giulio F. Draetta. DPY30 loss leads to DNA re-replication and immunoediting in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 303.

Abstract 877: Protein-protein interactions in cancer: An in-silico analysis of Cdc6 interactions

Abstract Aberrant activity of cell cycle proteins is a hallmark of cancer and therefore, cell cycle regulators are considered attractive targets in cancer therapy. One such regulator, Cell division cycle 6 (Cdc6), is a key conserved cell cycle protein responsible for the initiation of eukaryotic DNA replication. Cdc6 overexpression has been observed as an early event in malignancies. Additionally, Cdc6 has also been implicated in contributing to the aggressiveness of cervical and bladder cancers. In budding yeast, Cdc6 collaborates with proteins such as Cyclin B2 (Clb2) and Cell division control protein 4 (Cdc4) to ensure the fidelity of cell division by preventing re-replication. The perfectly orchestrated sequence of events requires Cdc6 to interact with other cell cycle proteins during the correct stage of the cell cycle. During S-phase, Cyclin-dependent kinase 1 (Cdk1) regulates Cdc6 levels and Cdc6 is targeted for degradation by polyubiquination via Cdc4 (SCF-complex). In mitosis, phosphorylation by Cdk1, induces the binding of Cdc6 to Clb2-Cdk1. Clb2 binds to Cdc6’s 44PEKLQF49 motif at the Cdc6 N-terminus thereby preventing Cdc6 from licensing origins, preventing DNA re-replication, and ensuring genomic integrity. Although, it has been well studied how each Cdc6 phosphorylation site regulates protein binding, in the absence of full-length structures for many of these proteins, the interaction modes and molecular details for Cdc6’s interaction with its protein partners remain unknown. In this study, we have characterized and analyzed Cdc6’s interactions with some of its key interactors. We present a robust computational approach to understand the molecular mechanisms of Cdc6 interactions with Cdc4 and Clb2, which includes homology/ab initio modeling of the full-length structures including their intrinsically disordered regions (IDRs), and predicted interaction scenarios. Our results show that the Cdc6-Cdc4 interaction displays a dynamic shift between S-phase and mitosis. In S-phase, Cdc4 binds to the N’ phosphodegrons on Cdc6, but in mitosis, Cdc4 binding shifts to the C’ phosphodegrons. In addition, we show Clb2’s K270, within its hydrophobic patch, binds E45 in Cdc6’s 44PEKLQF49 motif. We also provide a comparison of Clb2’s interaction with Cdc6 with that of Bud3 to assess the conservation of the binding mechanism. We show that the molecular mechanism is conserved with similar residues and motifs being involved in the interaction. By exploring the molecular details underlying these protein-protein interactions, we can gain insightful information and identify interface residues that are responsible for specific protein-protein interactions. Such molecular details are critical to pave the way to the discovery of druggable targets and ultimately lead to the development of new cancer therapeutics. Citation Format: Andriele Silva Eichner, Jasmin Philip, Amy Ikui, Shaneen Singh. Protein-protein interactions in cancer: An in-silico analysis of Cdc6 interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 877.

CDT1 is the major functional regulatory subunit of the pre-replication complex in zygotes.

Pre-replication complex (pre-RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre-RC, and proper regulation of CDT1 and MCM2 are necessary for DNA replication and cell proliferation. The present study aimed to explore the role of CDT1 and MCM2 in oocyte meiotic maturation and early embryonic development. The depletion and overexpression of Cdt1 and Mcm2 in oocyte and zygote were achieved by microinjecting specific siRNA and mRNA to explored their functions in oocyte meiotic maturation and embryonic development. Then, we examined the effect of CDT1 and MCM2 on other signal pathways by immunostaining the expression of related maker genes. We showed that neither depletion nor overexpression of Cdt1 affected oocyte meiotic progressions. The CDT1 was degraded in S phase and remained at a low level in G2 phase of zygote. Exogenous expression of Cdt1 in G2 phase led to embryo attest at zygote stage. Mechanistically, CDT1 overexpression induced DNA re-replication and thus DNA damage check-point activation. Protein abundance of MCM2 was stable throughout the cell cycle, and embryos with overexpressed MCM2 could develop to blastocysts normally. Overexpression or depletion of Mcm2 also had no effect on oocyte meiotic maturation. Our results indicate that pre-RC subunits CDT1 and MCM2 are not involved in oocyte meiotic maturation. In zygote, CDT1 but not MCM2 is the major regulator of DNA replication in a cell cycle dependent manner. Furthermore, its' degradation is essential for zygotes to prevent from DNA re-replication in G2 stage.

Injury-Induced DNA Re-Replication Leads to Tubular Cell Polyploidization in FAN1-Deficient Kidneys

Injury-Induced DNA Re-Replication Leads to Tubular Cell Polyploidization in FAN1-Deficient Kidneys

Transposon activity, local duplications and propagation of structural variants across haplotypes drive the evolution of the Drosophila S2 cell line

BackgroundImmortalized cell lines are widely used model systems whose genomes are often highly rearranged and polyploid. However, their genome structure is seldom deciphered and is thus not accounted for during analyses. We therefore used linked short- and long-read sequencing to perform haplotype-level reconstruction of the genome of a Drosophila melanogaster cell line (S2-DRSC) with a complex genome structure.ResultsUsing a custom implementation (that is designed to use ultra-long reads in complex genomes with nested rearrangements) to call structural variants (SVs), we found that the most common SV was repetitive sequence insertion or deletion (> 80% of SVs), with Gypsy retrotransposon insertions dominating. The second most common SV was local sequence duplication. SNPs and other SVs were rarer, but several large chromosomal translocations and mitochondrial genome insertions were observed. Haplotypes were highly similar at the nucleotide level but structurally very different. Insertion SVs existed at various haplotype frequencies and were unlinked on chromosomes, demonstrating that haplotypes have different structures and suggesting the existence of a mechanism that allows SVs to propagate across haplotypes. Finally, using public short-read data, we found that transposable element insertions and local duplications are common in other D. melanogaster cell lines.ConclusionsThe S2-DRSC cell line evolved through retrotransposon activity and vast local sequence duplications, that we hypothesize were the products of DNA re-replication events. Additionally, mutations can propagate across haplotypes (possibly explained by mitotic recombination), which enables fine-tuning of mutational impact and prevents accumulation of deleterious events, an inherent problem of clonal reproduction. We conclude that traditional linear homozygous genome representation conceals the complexity when dealing with rearranged and heterozygous clonal cells.

A mechanism of origin licensing control through autoinhibition of S. cerevisiae ORC\xb7DNA\xb7Cdc6

The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5–2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.

CRL4Cdt2 Ubiquitin Ligase, A Genome Caretaker Controlled by Cdt2 Binding to PCNA and DNA.

The ubiquitin ligase CRL4Cdt2 plays a vital role in preserving genomic integrity by regulating essential proteins during S phase and after DNA damage. Deregulation of CRL4Cdt2 during the cell cycle can cause DNA re-replication, which correlates with malignant transformation and tumor growth. CRL4Cdt2 regulates a broad spectrum of cell cycle substrates for ubiquitination and proteolysis, including Cdc10-dependent transcript 1 or Chromatin licensing and DNA replication factor 1 (Cdt1), histone H4K20 mono-methyltransferase (Set8) and cyclin-dependent kinase inhibitor 1 (p21), which regulate DNA replication. However, the mechanism it operates via its substrate receptor, Cdc10-dependent transcript 2 (Cdt2), is not fully understood. This review describes the essential features of the N-terminal and C-terminal parts of Cdt2 that regulate CRL4 ubiquitination activity, including the substrate recognition domain, intrinsically disordered region (IDR), phosphorylation sites, the PCNA-interacting protein-box (PIP) box motif and the DNA binding domain. Drugs targeting these specific domains of Cdt2 could have potential for the treatment of cancer.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

Anatomy and evolution of a DNA replication origin.

DNA amplification occurs at the DNA puff II/9A locus in the fungus fly Sciara coprophila. As a foundation to study the molecular mechanism for the initiating events of II/9A DNA re-replication, we have sequenced 14kb spanning a DNase hypersensitive site (DHS) upstream of the 1kb amplification origin and through transcription units II/9-1 and II/9-2 downstream of the origin. These elements are annotated as well as the ORC binding site at the origin and the transition point (TP) between continuous and discontinuous DNA syntheses that marks the origin of bidirectional replication at the nucleotide level. A 9bp motif found at the TP is repeated near the other end of the 1 kb ORI and may identify a putative second TP. The steroid hormone ecdysone induces DNA amplification as well as transcription and puffing at locus II/9A. Within the 14 kb, several matches to the ecdysone response element (EcRE) consensus sequence were identified, including some in the amplification origin region. EcRE O-P is at a central axis of a remarkable symmetry, equidistant to the TPs that are themselves equidistant to EcRE O-1 and EcRE O-2. DNA sequence alterations have occurred throughout the II/9A region in a newly discovered polymorphism (#2). Polymorphism #2 is not specific to developmental stage, sex, or tissue, and it does not impair DNA amplification. The DHS, both 9bp TP sequences, and EcREs O-1, O-P, and O-2 are conserved between the polymorphism #1 and #2 sequences, suggesting their functional importance and retention during evolutionary selection. Moreover, a 72 bp sequence in the Sciara DHS at DNA puff II/9A is conserved in DNA puff C-3 of Rhynchosciara americana. Comparisons are discussed between the Sciara II/9A amplicon and the chorion locus amplicon on the third chromosome of Drosophila.

MLN4924 inhibits cell proliferation by targeting the activated neddylation pathway in endometrial carcinoma.

ObjectiveTo explore the neddylation pathway, found to be highly activated in various cancers, as a potential therapeutic target in endometrial carcinoma, one of the three most frequent malignant tumours in the female reproductive system.MethodsData from The Cancer Genome Atlas were analysed using online servers. Expression levels of key neddylation genes were validated by reverse-transcription polymerase chain reaction and western blots of tumour and adjacent tissues. Underlying mechanisms and the effects on cell activities of the neddylation pathway-specific inhibitor, MLN4924, were investigated in endometrial cancer cell lines.ResultsKey neddylation enzymes, ubiquitin conjugating enzyme E2 M (UBC12), ubiquitin conjugating enzyme E2 F (UBE2F), ring-box 1 (RBX1) and ring finger protein 7 (RBX2), were significantly overexpressed in endometrial carcinoma tissues versus normal tissues, but only UBE2F and RBX2 positively correlated with patient survival. MLN4924 significantly suppressed proliferation and colony formation in EC cells by inducing DNA re-replication, cell cycle arrest and apoptosis. Mechanism study revealed that MLN4924 induced the accumulation of cullin-RING ligase substrates in vitro.ConclusionsThe neddylation pathway was identified to play an important role in endometrial cancer. The neddylation specific inhibitor, MLN4924, may be a potential therapeutic drug for endometrial carcinoma.

Regulation of DNA Replication Licensing and Re-Replication by Cdt1.

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 CRL4Cdt2 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 CRL4Cdt2 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 CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 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.

In silicoanalysis of DNA re-replication across a complete genome reveals cell-to-cell heterogeneity and genome plasticity

DNA replication is a complex and remarkably robust process: despite its inherent uncertainty, manifested through stochastic replication timing at a single-cell level, multiple control mechanisms ensure its accurate and timely completion across a population. Disruptions in these mechanisms lead to DNA re-replication, closely connected to genomic instability and oncogenesis. Here, we present a stochastic hybrid model of DNA re-replication that accurately portrays the interplay between discrete dynamics, continuous dynamics and uncertainty. Using experimental data on the fission yeast genome, model simulations show how different regions respond to re-replication and permit insight into the key mechanisms affecting re-replication dynamics. Simulated and experimental population-level profiles exhibit a good correlation along the genome, robust to model parameters, validating our approach. At a single-cell level, copy numbers of individual loci are affected by intrinsic properties of each locus, in cis effects from adjoining loci and in trans effects from distant loci. In silico analysis and single-cell imaging reveal that cell-to-cell heterogeneity is inherent in re-replication and can lead to genome plasticity and a plethora of genotypic variations.

Importance of Cullin4 Ubiquitin Ligase in Malignant Pleural Mesothelioma

Simple SummaryMalignant pleural mesothelioma (MPM) is an incurable and aggressive malignancy mainly caused by exposure to asbestos fibers. Survival outcomes following the standard of care treatment, including chemotherapy and surgery, remain dismal. Targeted treatments are shortcomings for MPM, as this tumor is driven primarily by a loss of tumor suppressor genes. In this study, we explored the importance of proteins that have been shown to be dysregulated in MPM as a consequence of tumor suppressor gene loss, CUL4A and CUL4B. We assessed their expression levels and identified their correlation with clinical outcomes of MPM. We also aimed to test the efficacy and mechanisms of the available treatments that target these proteins, pevonedistat using in vitro and in vivo models. Our data suggested that CUL4B might serve as a treatment target for MPM and revealed novel mechanism of pevonedistat in the MPM tumor microenvironment. This data may be useful for understanding its efficacy in patients from clinical trials. Neurofibromatosis type 2 (NF2), the tumor suppressor frequently lost in malignant pleural mesothelioma (MPM), suppresses tumorigenesis in part by inhibiting the Cullin4 ubiquitin ligase (CUL4) complex in the nucleus. Here, we evaluated the importance of CUL4 in MPM progression and tested the efficacy of cullin inhibition by pevonedistat, a small molecule inhibiting cullin neddylation. CUL4 paralogs (CUL4A and CUL4B) were upregulated in MPM tumor specimens compared to nonmalignant pleural tissues. High gene and protein expressions of CUL4B was associated with a worse progression-free survival of MPM patients. Among 13 MPM cell lines tested, five (38%) were highly sensitive to pevonedistat (half maximal inhibitory concentration of cell survival IC50 < 0.5 µM). This remained true in a 3D spheroid culture. Pevonedistat treatment caused the accumulation of CDT1 and p21 in both sensitive and resistant cell lines. However, the treatment induced S/G2 cell cycle arrest and DNA rereplication predominantly in the sensitive cell lines. In an in vivo mouse model, the pevonedistat treatment significantly prolonged the survival of mice bearing both sensitive and resistant MPM tumors. Pevonedistat treatment reduced growth in sensitive tumors but increased apoptosis in resistant tumors. The mechanism in the resistant tumor model may be mediated by reduced macrophage infiltration, resulting from the suppression of macrophage chemotactic cytokines, C-C motif chemokine ligand 2 (CCL2), expression in tumor cells.

Loss of hepatocyte cell division leads to liver inflammation and fibrosis.

The liver possesses a remarkable regenerative capacity based partly on the ability of hepatocytes to re-enter the cell cycle and divide to replace damaged cells. This capability is substantially reduced upon chronic damage, but it is not clear if this is a cause or consequence of liver disease. Here, we investigate whether blocking hepatocyte division using two different mouse models affects physiology as well as clinical liver manifestations like fibrosis and inflammation. We find that in P14 Cdk1Liv-/- mice, where the division of hepatocytes is abolished, polyploidy, DNA damage, and increased p53 signaling are prevalent. Cdk1Liv-/- mice display classical markers of liver damage two weeks after birth, including elevated ALT, ALP, and bilirubin levels, despite the lack of exogenous liver injury. Inflammation was further studied using cytokine arrays, unveiling elevated levels of CCL2, TIMP1, CXCL10, and IL1-Rn in Cdk1Liv-/- liver, which resulted in increased numbers of monocytes. Ablation of CDK2-dependent DNA re-replication and polyploidy in Cdk1Liv-/- mice reversed most of these phenotypes. Overall, our data indicate that blocking hepatocyte division induces biological processes driving the onset of the disease phenotype. It suggests that the decrease in hepatocyte division observed in liver disease may not only be a consequence of fibrosis and inflammation, but also a pathological cue.

Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

Early Mitotic Inhibitor 1 Regulates DNA Re-replication Mediated by Human Papillomavirus Subtype 16 E7 in Response to DNA Damage

Objective To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Methods Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Results Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(t=6.397,P=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(t=8.241,P=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(t=2.916,P=0.0434;t=3.452,P=0.0260). Conclusion In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.

Quinacrine Based Gold Hybrid Nanoparticles Caused Apoptosis through Modulating Replication Fork in Oral Cancer Stem Cells.

The presence of cancer stem cells (CSCs) in the tumor microenvironment is responsible for the development of chemoresistance and recurrence of cancer. Our previous investigation revealed the anticancer mechanism of quinacrine-based silver and gold hybrid nanoparticles (QAgNP and QAuNP) in oral cancer cells, but to avoid cancer recurrence, it is important to study the effect of these nanoparticles (NPs) on CSCs. Here, we developed an in vitro CSCs model using SCC-9 oral cancer cells and validated via FACS analysis. Then, 40-60% of cells were found to be CD44+/CD133+ and CD24-. QAuNP showed excellent anti-CSC growth potential against SCC-9-cancer stem like cells (IC50 = 0.4 μg/mL) with the down-regulation of representative CSC markers. Prolonged exposure of QAuNP induced the S-phase arrest and caused re-replication shown by the extended G2/M population and apoptosis to SCC-9-CSC like cells. Up-regulation of BAX, PARP cleavage, and simultaneous down-regulation of Bcl-xL in prolonged treatment to CSCs suggested that the majority of the cells have undergone apoptosis. QAuNP treatment also caused a loss in DNA repair in CSCs. Mostly, the base excision repair (BER) components (Fen-1, DNA ligase-1, Pol-β, RPA, etc.) were significantly down-regulated after QAuNP treatment, which suggested its action against DNA repair machinery. The replication fork maintenance-related proteins, RAD 51 and BRCA-2, were also deregulated. Very surprisingly, depletion of WRN (an interacting partner for Pre-RC and Fen-1) and a significant increase in expression of fork-degrading nuclease MRE-11 in 96 h treated NPs were observed. Results suggest QAuNP treatment caused excessive DNA damage and re-replication mediated replication stress (RS) and stalling of the replication fork. Inhibition of BER components hinders the flap clearance activity of Fen-1, and it further caused RS and stopped DNA synthesis. Overall, QAuNP treatment led to irreparable replication fork movement, and the stalled replication fork might have degraded by MRE-11, which ultimately results in apoptosis and the death of the CSCs.