DNA Licensing Research Articles

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.

MCM5 is a Novel Therapeutic Target for Glioblastoma.

MCM5 is a DNA licensing factor involved in cell proliferation and has been previously established as an excellent biomarker in a number of malignancies. Nevertheless, the role of MCM5 in GBM has not been fully clarified. The present study aimed to investigate the potential roles of MCM5 in the treatment of GBM and to elucidate its underlying mechanism, which is beneficial for developing new therapeutic strategies and predicting prognosis. Firstly, we obtained transcriptomic and proteomic data from the TCGA and CPTAC databases on glioma patients. Employing the DeSeq2 R package, we then identified genes with joint differential expression in GBM tissues subjected to chemotherapy. To develop a prognostic risk score model, we performed univariate and multivariate Cox regression analyses. In vitro knockdown and overexpression of MCM5 were used to further investigate the biological functions of GBM cells. Additionally, we also delved into the upstream regulation of MCM5, revealing associations with several transcription factors. Finally, we investigated differences in immune cell infiltration and drug sensitivity across diverse risk groups identified in the prognostic risk model. In this study, the chemotherapy-treated GBM samples exhibited consistent alterations in 46 upregulated and 94 downregulated genes at both the mRNA and protein levels. Notably, MCM5 emerged as a gene with prognostic significance as well as potential therapeutic relevance. In vitro experiments subsequently validated the role of increased MCM5 expression in promoting GBM cell proliferation and resistance to TMZ. Correlations with transcription factors such as CREB1, CTCF, NFYB, NRF1, PBX1, TEAD1, and USF1 were discovered during upstream regulatory analysis, enriching our understanding of MCM5 regulatory mechanisms. The study additionally delves into immune cell infiltration and drug sensitivity, providing valuable insights for personalized treatment approaches. This study identifies MCM5 as a key player in GBM, demonstrating its prognostic significance and potential therapeutic relevance by elucidating its role in promoting cell proliferation and resistance to chemotherapy.

Abstract P1-13-05: Extracellular vesicles-transported lncRNA BCDR1 promotes tumor cell proliferation and therapy resistance via upregulating G1/S-phase transition in breast cancer

Abstract Background Extracellular vesicles (EVs), secreted by tumor cells for intercellular communication, play an important role in breast cancer progression. Previous studies have proved that lncRNA could use EVs to transmit signals and affect the progression and treatment of breast cancer. In addition, a previous study has found that, in HER2 therapy-resistant tumors, CDK4/6 activity and cell cycle progression genes were involved and might serve as treatment targets. However, recent clinical trials were not supportive of the use of CDK4/6 inhibitors in HER2 enriched internal subtypes of breast cancer. In this study, we aim to explore the role of EVs-transported long noncoding RNA BCDR1 in the promotion of cell cycle progression in HER2-positive breast cancer, exploring a new potential target in combination with CDK4/6 inhibitor and anti-HER2 treatment. Methods Samples were collected from patients with HER2 overexpression receiving neoadjuvant therapy. RNA-seq was performed to identify differentially expressed RNAs between pathological complete response (pCR) and non-pCR group. In addition, serum extracellular vesicles were collected and determined through RNA-seq. The drug response and proliferation rate of tumor cells were measured in breast cancer cells (HCC-1954, BT-474 and MCF-7). Quantitative RT-PCR (qRT-PCR) was used to detect the expression levels of BCDR1 and its potential target genes. RNA-seq and GSEA analysis was carried out to determine the target pathways regulated by BCDR1. DNA fiber assay, mass spectrometry, and flow cytometry were used to understand the underlying mechanism of BCDR1. Results Core needle biopsy tissues from HER2-positive breast cancer patients with any ER status were collected before NAC. Among genes differentially overexpressed, BCDR1 was found to be downregulated in pCR group compared with non-pCR group. BCDR1 was also elevated in plasma EVs in the non-pCR group. In addition, overexpression of BCDR1 in breast cancer cells promoted cell proliferation and HER2 treatment resistance. Through pathway analysis, we found BCDR1 could facilitate G1/S-phase transition. Flow cytometry confirmed these findings. We also noticed that proteins that regulate DNA licensing including minichromosome maintenance proteins (MCMs) were enriched in BCDR1 overexpression cells. Through DNA fiber assay, we confirmed that BCDR1 could promote DNA replication initiation. Interestingly, under CDK4/6 inhibitor treatment, BCDR1 was induced, unrevealing an internal treatment-resistant mechanism in these tumor cells. Through EVs RT-qPCR, we found BCDR1 could transport from high expression cells to low, with the same biological function. We also explored antisense oligonucleotide (ASO) use in the inhibition of BCDR1 biological function and transportation. Conclusion BCDR1 promotes cell proliferation and therapy resistance via upregulating DNA licensing in breast cancer. BCDR1 could transported through EVs with same biological function. This study also suggests that BCDR1 could serve as a biomarker and therapeutic target in breast cancer. Citation Format: Qi Zhang, Bingqiu Xiu, Yayun Chi, Jiong Wu. Extracellular vesicles-transported lncRNA BCDR1 promotes tumor cell proliferation and therapy resistance via upregulating G1/S-phase transition in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-05.

P723: LOSS OF THE NK MOTIF AND ANKYRIN REPEAT DOMAIN 1 (KANK1) LEADS TO LYMPHOID COMPARTMENT DYSREGUATION IN MICE

Background: We identified a novel germline loss of heterozygosity (LOH) mutation encompassing the NK Motif And AnKyrin Repeat Domain 1 (KANK1) gene in a young Myelodysplastic syndrome (MDS) patient and his healthy father, with no additional MDS-related mutations or chromosomal abnormalities. Clinical investigation suggested development of MDS following severe infection, likely triggered by an autoimmune mechanism. KANK1 protein plays a role in the control of cytoskeleton formation by regulating actin polymerization. A t(5;9) translocation resulting in a fusion of the platelet-derived growth factor receptor beta gene (PDGFRB) and KANK1 was detected in a patient with myeloid neoplasm (MN) characterized with severe thrombocythemia. The protein is thought to have a tumour suppressor function, as its expression is downregulated or missing in several tumor tissues, while over-expressing the protein was reported to inhibit the proliferation of tumor cells in solid cancer models. In addition, defects in cytoskeletal proteins are reported to contribute to immunological dysfunction and disruption of normal immune processes. Aims: To investigate the role of KANK1 in the regulation of normal hematopoiesis and the development of myeloid disorders. Methods: We generated a new genetically modified KANK1 (Kank1-/-) knockout mouse model and analysed the phenotypic and developmental changes in hematopoiesis in young and aged mice. Results: We confirmed the loss of Kank1 mRNA and protein expression by q-PCR and targeted protein mass-spectrometry respectively. The mice are viable and fertile, and reproduced according to mendelian ratio. Over the two years observation period, Kank1-/- mice did not exhibit any signs of disease, and their peripheral blood parameters remained within the normal range. Flow cytometry analysis of bone marrow (BM) cells from 17-20 weeks kank1-/- old mice showed minor increase in CD3+ T-cell population, while the expression of the double positive B220/CD19 B-cells population was reduced in Kank1-/- compared to Kank1+/+. Comprehensive flow cytometry analysis of spleen and thymus cells from young (n=8 each, 6-7 weeks old) and aged (n=8, 68-71 weeks old) Kank1-/- mice showed further dysregulation of the lymphoid compartment when compared to Kank1+/+. In young mice, a slight decrease of Treg cells is noticed in the spleen, coupled with a slight decrease of activated CD4 in the peripheral blood. In the spleen of aged Kank1-/- mice, the proportions of macrophages and Treg cells were decreased, while the central memory CD4+ T-cells were increased. Total protein analysis from BM cells of Kank1-/- (n=3) compared to Kank1+/+ mice (n=3) showed a significant reduction (FC >0.5, adj.p. <0.05) in proteins associated with cytoskeleton formation such as Myosin1 (MYH1), Tropomysin-1 (TDM1) and Annexin (ANXA2), and extracellular matrix components such as Collagena(CO1A1) and Matrix metalloproteinase9 (MMP9). We also detected a significant increase in expression of the DNA licensing factor minichromosome maintenance complex component 7 (MCM7), and the mitochondrial membrane associated transporter ATP binding Cassette Subfamily B Member 10 (ABCBA). Summary/Conclusion: Dysregulation of the immune system has been widely recognized to play a role in the development of MDS. Based on our results, we propose a role for KANK1 in regulating the development of immune cells in the mouse through it invovlement in cytoskeleton regulation. In vivo infectious challenge experiments using our model will futher elucidate the importance of KANK1 in the development of MDS.

Spatial and temporal resolution of mORC4 fluorescent variants reveals structural requirements for achieving higher order self-association and pronuclei entry

The Origin Replication Complex (ORC), which is a multi-subunit protein complex composed of six proteins ORC1–6, is essential for initiating licensing at DNA replication origins. We have previously reported that ORC4 has an alternative function wherein it forms a cage surrounding the extruded chromatin in female meiosis and is required for polar body extrusion (PBE). As this is a highly unexpected finding for protein that normally binds DNA, we tested whether ORC4 can actually form larger, higher order structures, which would be necessary to form a cage-like structure. We generated two fluorescent constructs of mouse ORC4, mORC4-EGFP and mORC4-FlAsH, to examine its spatial dynamics during oocyte activation in live cells. We show that both constructs were primarily monomeric throughout the embryo but self-association into larger units was detected with both probes. However, mORC4-FlAsH clearly showed higher order self-association and unique spatial distribution while mORC4-EGFP failed to form large structures during Anaphase II. Interestingly, both variants were found in the pronuclei suggesting that its role in DNA licensing is still functional. Our results with both constructs support the prediction that ORC4 can form higher order structures in the cytoplasm, suggesting that it is possible to form a cage-like structure. The finding that FlAsH labeled ORC4 formed demonstrably larger higher order structures than ORC4-GFP suggests that ORC4 oligomerization is sensitive to the bulky addition of GFP at its carboxy terminus.

Abstract 3466: Growth inhibitory MEK-ERK signaling in the intestinal epithelium

Abstract Increasing evidence points to members of the PKC family of serine/threonine kinases as important tumor suppressors; however, the signaling pathways involved remain poorly defined. Our previous studies have determined that PKCα, which acts as a tumor suppressor in the epithelium of the intestine/colon, is activated coincident with growth arrest in intestinal crypts in vivo. PKCα activity induces cell cycle withdrawal in intestinal epithelial cells, a process that involves downregulation of cyclin D1 and Id1, upregulation of the cyclin dependent kinase inhibitors p21cip1 and p27kip1, and characteristic changes in pocket proteins and DNA licensing factors. Pharmacological inhibitors of the ERK pathway revealed that these changes are dependent on activation of MEK-ERK signaling, thus identifying a growth inhibitory ERK-mediated signaling axis in the intestinal epithelium. Given the interest in inhibitors of the ERK pathway for treatment of colon and other cancers, characterization of this tumor suppressive pathway will be essential for rational development of this treatment modality and to avoid and/or manage potential adverse effects of ERK inhibition in patients. To this end, the mechanistic basis for the growth suppressive effects of the PKCα→ERK module on cyclin D1, Id1 and p21cip1 are being investigated in non-transformed intestinal epithelial cells and colon cancer cells. Use of selective inhibitors and siRNA-mediated knockdown has determined that PKCα activates multiple components of the ERK pathway, including RAS, RAF, MEK and ERK. Notably, tumor suppressive effects are seen in colon cancer cells that harbor activating mutations in KRAS, indicating that growth inhibitory ERK signaling can override oncogenic signals arising from RAS mutation. PKCα signaling is associated with activation of ARAF, BRAF and CRAF and with induction of RAF heterodimers. A requirement for RAF activity was confirmed by the ability of LY3009120 or sorafenib to block growth inhibitory effects of PKCα. However, knockdown of individual RAF proteins fails to block these effects of PKCα, indicating some redundancy between the RAF proteins in mediating growth inhibitory signals. While the scaffolding protein KSR1 has been shown to be required for oncogenic RAS-ERK signaling, PKCα was still able to elicit growth inhibitory effects in KSR1 knockdown HCT-116 colon cancer cells. These data indicate that differential use of scaffolding proteins may explain both the growth inhibitory effects of PKCα-ERK signaling and their ability to override oncogenic RAS signals. A better understanding of these mechanisms may serve to guide the use of ERK inhibitors for colon cancer therapy. Supported by NIH grants DK60632, CA54807, CA157774 and CA191894. Citation Format: Navneet Kaur, Robert Lewis, Adrian Black, Jennifer Black. Growth inhibitory MEK-ERK signaling in the intestinal epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3466.

DNA replicationmachinery is required for development inDrosophila.

In Drosophila, some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

Abstract 5495: GANT61-induced cell death involves inhibition of transcription and DNA licensing at GLI sites in the promoters of GLI-dependent target genes in human colon carcinoma cells

Abstract The GLI genes, GLI1 and GLI2, are transcriptional regulators of the Hedgehog signaling response, binding at promoters to GACCACCCA-like consensus sequences. From genetic and biochemical studies, GLI2 is the primary mediator of HH signaling, which activates GLI1 to transcriptionally regulate target genes. In cancers both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated by oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer. GANT61, a specific GLI inhibitor, induced extensive cytotoxicity in human models of colon cancer, indicating GLI to be a critical target in cancer cell survival. We have determined FOXM1 to be a transcriptional target of GLI1 at a GCCCACCCA consensus sequence. In HT29 cells, inhibition of GLI binding to the FOXM1 promoter by GANT61 led to inhibition of binding of the pause-release factors DSIF, NELF, and p-TEFb in the region of the TSS, with inhibition of binding of Pol II at both GLI and TSS sites. In R-loop regions, RNA:DNA hybrid formation was reduced at both the GLI and TSS sites in the FOXM1 promoter by GANT61, sensitive to RNaseH. Bisulfite conversion/PCR also determined &amp;gt;50% reduction in ssDNA (C-&amp;gt;T conversions) in the GLI binding region in GANT61-treated cells. Data support GANT61-induced inhibition of GLI-dependent transcription at the PIC before R-loops are formed. Pretreatment of HT29 cells with α-amanitin (Pol II inhibitor) reduced GANT61-induced γH2AX foci, indicating the importance of transcription in GANT61-induced DNA damage. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication were linked. By co-immunoprecipitation, GLI1 bound the DNA replication licensing factors ORC4, CDT1, and MCM2, decreased in the presence of GANT61. By confocal microscopy, significant co-localization of GLI1 and ORC4 foci was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. In addition to non-transcriptional mechanisms of interaction between GLI1 and DNA licensing factors, CDT1 was found to be a transcription target of GLI1 at a putative GACCACCCG consensus sequence in the promoter. ChIP analysis determined significant enrichment of γH2AX at this site in GANT61-treated HT29 cells. Overexpression of CDT1 in HT29 cells reduced GANT61-induced cell death and cleavage of caspase-3, indicating a significant role of DNA replication licensing in the mechanism of induction of GANT61-induced cell death. In summary, we have demonstrated inhibition of GLI-dependent transcription by GANT61 at the PIC during the initiation of RNA synthesis, and the importance of inhibiting DNA replication licensing at sites in promoter regions that bind the transcription factor GLI1 by both non-transcriptional and transcriptional mechanisms. Supported by NCI Award 1 RO1 CA183921-01A1 and by Southern Research Institute Citation Format: Ruowen Zhang, Jiahui Wu, Sylvain Ferrandon, Katie J. Glowacki, Janet A. Houghton. GANT61-induced cell death involves inhibition of transcription and DNA licensing at GLI sites in the promoters of GLI-dependent target genes in human colon carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2017-5495

Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing

The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

P16 Gene Transfer Induces Centrosome Amplification and Abnormal Nucleation Associated with Survivin Downregulation in Glioma Cells

Objective: In human glioma cells, p16 gene transfer induced G1/S arrest, increased radiosensitivity and abnormal nucleation (especially bi- and multinucleation). Survivin suppression caused G2/M arrest, radiosensitization and an increase in aneuploidy accompanied by centrosome amplification. Abnormal nucleation and aneuploidy represent chromosome instability (CIN), and it is well known that centrosome amplification leads to CIN. However, little has been reported that suggests that transferring p16 causes centrosome overduplication during the G1/S phase. Methods: The p16 gene was transferred into p16-null human glioma cell lines (U251MG and D54MG) using adenovirus with or without irradiation. Centrosome amplification was evaluated by immunofluorescence. We also investigated the DNA replication licensing factor CDT1, its inhibitor geminin and survivin expression as regulators of chromosomal segregation. Results: p16 gene transfer with radiation initiated the greatest degree of centrosome overduplication. CDT1 showed low levels, geminin was unchanged and survivin decreased in Ax-hp16-infected cells with radiation. Those changes of factors affecting DNA licensing or chromosomal segregation might contribute to CIN. Conclusion: p16 transfer caused centrosome amplification even in G1/S phase-arrested cells. This suggests that p16 is involved in abnormal nucleation and radiosensitization in human glioma cells.

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function.

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency.

A spontaneous Cdt1 mutation in 129 mouse strains reveals a regulatory domain restraining replication licensing

Cdt1 is required for loading the replicative DNA helicase MCM2/7, a process known as DNA replication licensing. Here we show that 129 mouse strains express a Cdt1 mutated allele with enhanced licensing activity. The mutation, named Δ(6)PEST, involves a six-amino acid deletion within a previously uncharacterized PEST-like domain. Cdt1 Δ(6)PEST and more extensive deletions exhibit increased re-replication and transformation activities that are independent of the Geminin and E3 ligase pathways. This PEST domain negatively regulates cell cycle-dependent chromatin recruitment of Cdt1 in G2/M phases of the cell cycle. Mass spectrometry analysis indicates that Cdt1 is phosphorylated at sites within the deleted PEST domain during mitosis. This study reveals a conserved new regulatory Cdt1 domain crucial for proper DNA licensing activity and suggests a mechanism by which the presence of Cdt1 in G2/M phases does not lead to premature origin licensing. These results also question the usage of 129 mouse strains for knockout analyses.

An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.

Inhibition of the CRBN-DDB1-CUL4-ROC1 E3 Ubiquitin Ligase Mediates the Anti-Proliferative and Immunomodulatory Properties of Lenalidomide

Abstract 919Lenalidomide is a highly effective drug for the treatment of del(5q) MDS and multiple myeloma, and its use in a range of other conditions is being actively explored. Despite its increasing use for the treatment of malignancies, the precise mechanism of action of lenalidomide has not been established. We sought to identify the direct protein targets of lenalidomide using a quantitative, mass spectrometry-based proteomic approach we developed.Using a validated derivative of lenalidomide immobilized to beads, we identified DDB1 as a target of the drug by affinity enrichment of protein binders and analysis by high performance LC-MS/MS. DDB1, together with CRBN, CUL4A, and ROC1, forms an E3 ubiquitin ligase known as CRBN-CRL4. We confirmed that members of the complex bind to the immobilized lenalidomide derivative, and could be competed off with soluble lenalidomide, further supporting the role of CRBN-CRL4 in the actions of lenalidomide.CRL4 targets multiple proteins for ubiquitination and subsequent proteasomal degradation, including the cell cycle regulators CDKN1A (p21) and CDKN1B (p27), as well as the DNA licensing factor CDT1. We hypothesized that lenalidomide disrupts the ubiquitination of these and other proteins, leading to increased levels of the respective targets. We found that treatment of the lenalidomide sensitive cell line MM1S and NCI-H929 increased protein levels of p21, p27 and CDT1 in a dose and time dependent manner. Furthermore, overexpression of these three targets led to growth inhibition. Similarly, knockdown of DDB1, CUL4A, ROC1 and CRBN by lentiviral shRNAs increased p21 and p27 protein levels and inhibited growth of these cell lines.Lenalidomide is also known to increase IL-2, promote erythropoiesis and inhibit TNF-alpha. We found that in activated primary human T cells, shRNA knockdown of DDB1 recapitulated the stimulatory effects of lenalidomide on IL-2 expression levels and release. We also found that shRNA knockdown of DDB1 and CRBN recapitulated the pro-erythropoietic effects of the drug with an increase in the number of colony-forming units-erythroid (CFU-E) compared to control knockdown. Experiments studying the effects on TNF-alpha are underway.To further establish that the CRBN-CRL4 complex is the target of lenalidomide, we tested a previously published mutant form of CRBN which prevents binding of the drug to the complex. Ectopic expression of this mutant CRBN conferred resistance to lenalidomide induced cell death to multiple myeloma cells. It also resulted in the loss of CFU-E production by lenalidomide.To gain further insight into how lenalidomide might disrupt the function of the CRBN-CRL4 complex, we did immunoprecipitation against CRBN with or without the drug and found that lenalidomide disrupts the formation of the complex by preventing binding of ROC1, the adaptor protein to ubiquitin charged E2 conjugating enzyme. Using in vivo and in vitro ubiquitination assays, we also demonstrated that lenalidomide inhibits the auto-ubiquitination of CRBN. We are currently performing a ubiquitin profiling experiment to identify other protein targets that might be affected by the disruption of the CRBN-CRL4 complex by lenalidomide.Our study establishes that lenalidomide's antiproliferative and immunomodulatory properties rely on binding to CRBN-CRL4 and inhibiting its function as ubiquitin ligase. Ito et al. showed 2010 that the same mechanism is also responsible for the teratogenic effects of thalidomide. The characterization of lenalidomide as a specific E3 ubiquitin ligase inhibitor will provide insight into the mechanism of therapeutic efficacy in MDS and multiple myeloma, and serves as a proof-of-concept that selective ubiquitin ligases are efficacious targets for cancer therapy. Disclosures:No relevant conflicts of interest to declare.

Stain-Free technology as a normalization tool in Western blot analysis

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.

Regulation of DNA Replication Licensing

In eukaryotic cells, DNA replication is tightly regulated to occur only once per cell cycle. DNA licensing is a mechanism to guarantee this aim; that is, licensing of replication initiation is permitted during late M phase to G1 phase. The license is canceled by the start of DNA replication. Once DNA replication begins, the license is never given until the next late M phase. The licensing corresponds to the process of assembling components of the pre-replication complex (pre-RC) on the replication origin DNA. This pre-RC is the target of several different regulation systems to prevent rereplication of DNA during a single cell cycle. In this review, the regulation mechanisms mainly in mammals to control assembling components of the pre-RC will be discussed.

Licensing of DNA for Replication in Mammalian Zygotes Follows a Different Pathway Than Somatic Cells.

INTRODUCTION: DNA replication in the one-cell embryo has several unique features that differ from more frequently studied somatic cells. It is the only time the maternal and paternal genomes undergo different processes in the same cell. The life of the zygote begins with the maternal DNA completing meiosis and the paternal DNA being decondensed. Each genome is then housed within separate pronuclei and undergo DNA replication before eventually existing in the same nucleus in the two-cell stage. The initial differences in chromatin structure result in the two pronuclei developing asynchronously, with the maternal pronucleus forming slightly more rapidly than the paternal. Because of this, we tested whether the regulation of the initiation of DNA synthesis of the two pronuclei occurred simultaneously, or whether there are recognizable differences. Mammalian DNA replication origins must be licensed during G1 before DNA synthesis can begin. This ensures that all the DNA is replicated, and that each segment of DNA is only replicated once. Licensing requires that each replication origin interacts with a complex of proteins, referred to as the pre-replication complex (pre-RC). In somatic cells, the origin recognition complex (ORC1-6) mediates DNA-protein interactions by recruiting Cdc6, Cdt1 and the minichromosomal maintenance (MCM 2-7) proteins. We examined DNA replication licensing in the one cell embryo and found it was significantly different from that of somatic cells. METHODS: Mouse embryos were created by ICSI to ensure timing of development. Oocytes were parthenogenetically activated with SrCl2 for comparative analysis. The loading of ORC2 and MCM7 were monitored by immunocytochemistry through the two-cell stage. RESULTS: Neither ORC2 nor Mcm7 were detected in mature oocytes prior to fertilization. Surprisingly, the first licensing protein to bind chromatin in zygotes was MCM7, which first appeared on the maternal (and polar body) chromatin during anaphase II by 1hr after fertilization. At this same time point, ORC2 was clearly localized on the anaphase spindle between the separating chromosomes but was not detected on the chromosomes, themselves. Neither protein was visible in the paternal pronucleus. By 2 hrs after fertilization, both MCM7 and ORC2 were visible in the maternal pronucleus, but did not appear in the paternal pronucleus until three hrs after fertilization. During the second cell-cycle ORC2 appeared on the spindles and formed a ring structure surrounding the chromatin during anaphase while MCM7 loaded onto each set of DNA. Both proteins were present on the nuclei after 1hr into G1 of the second cell-cycle. CONCLUSIONS: Licensing of the maternal and paternal pronuclei appears to follow independent pathways with each pronucleus. This suggests licensing is relatively independent of cytoplasmic control. Both pronuclei appear to be fully licensed by 3 hrs after fertilization. While we could not differentiate the timing of MCM7 and ORC2 loading in the paternal pronucleus, it was clear that MCM7 bound the chromatin of the maternal pronucleus before ORC2. Current models for DNA licensing in somatic cells suggest that ORC2 binding is required for MCM7 loading. Our data suggest that either this is not the case in zygotes. Alternatively, ORC2 may bind first, but may not be a prerequisite for MCM7 binding in somatic cells. This research was supported by NIH grant HD060722. (platform)

The E3 ubiquitin ligase Cullin 4A regulates meiotic progression in mouse spermatogenesis

The Cullin-RING ubiquitin-ligase CRL4 controls cell cycle and DNA damage checkpoint response and ensures genomic integrity. Inactivation of the Cul4 component of the CRL4 E3 ligase complex in Caenorhabditis elegans by RNA interference results in massive mitotic DNA re-replication in the blast cells, largely due to failed degradation of the DNA licensing protein, CDT-1, and premature spermatogenesis. Here we show that inactivation of Cul4a by gene-targeting in mice only affected male but not female fertility. This male infertility phenotype resulted from a combination of decreased spermatozoa number, reduced sperm motility and defective acrosome formation. Agenesis of the mutant germ cells was accompanied by increased cell death in pachytene/diplotene cells with markedly elevated levels of phospho-p53 and CDT-1. Despite apparent normal assembly of synaptonemal complexes and DNA double strand break repair, dissociation of MLH1, a component of the late recombination nodule, was delayed in Cul4a−/− diplotene spermatocytes, which potentially led to subsequent disruptions in meiosis II and spermiogenesis. Together, our study revealed an indispensable role for Cul4a during male germ cell meiosis.

Abstract 1065: MicroRNA-1296 and genistein regulate DNA licensing proteins (MCM-genes) in prostate cancer

Abstract Introduction and Objective: The minichromosome maintenance (MCM) gene-family is essential for DNA replication and is found to be frequently up-regulated in a variety of cancers. In this study we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296, genistein and trichostatin A on the MCM-complex. Methods: Prostate cancer cell lines (LNCaP and PC3) were treated with different doses (0-50umol/L) of genistein (72 hours) and 100 ng/ml trichostatin-A (24 hours). Quantitative real-time PCR, immunohistochemical staining and Western analysis were performed to check mRNA and protein expression in cells and tissues. The small interfering RNA (siRNA) approach was employed to understand the functional significance of MCM2 gene. Fluorescence activated cell sorting (FACS) analysis was performed to monitor cell cycle distribution and apoptosis. PC3 cells were transfected with miR-1296 and miR-1296 inhibitor to investigate its effect on the MCM2 gene. Results: Profiling results showed that expression of MCM-genes was higher in prostate tumor sample and cell lines. Genistein and trichostatin A (TSA) significantly down-regulated expression of all MCM-genes. A specific siRNA was used to knock-down MCM2 mRNA and protein expression. FACS results showed that genistein, TSA and siRNA caused a significant decrease in the S-phase of the cell cycle. There was also down-regulation of CDT1, CDC7 and CDK2 genes which govern the loading of MCM-complex on chromatin. We found that microRNA-1296 targets the MCM2 mRNA sequence. MicroRNA-1296 expression is significantly down-regulated in prostate cancer compared to normal tissues. To investigate the relationship between MCM2 and miR-1296, a miR-1296 inhibitor was transfected into PC3 cells. Inhibition of miR-1296 up-regulated both MCM2 mRNA and protein, whereas over-expression caused a significant decrease in MCM2 mRNA, protein and the S phase of cell cycle. Conclusion: MCM-genes are excellent anti-cancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing an anti-MCM effect by miR-1296, genistein and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural non-toxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNAi is currently being implemented as a gene-specific approach for molecular medicine. By the same principle, the specific down-regulation of oncogenes by micro-RNA may contribute to novel therapeutic approaches in the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1065.