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Amin A, Wu R, Cheung MH, Scott JF, Wang Z, Zhou Z, Liu C, Zhu G, Wong CKC, Yu Z, Liang C. An Essential and Cell-Cycle-Dependent ORC Dimerization Cycle Regulates Eukaryotic Chromosomal DNA Replication. Cell Rep 2021; 30:3323-3338.e6. [PMID: 32160540 DOI: 10.1016/j.celrep.2020.02.046] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2018] [Revised: 10/04/2019] [Accepted: 02/10/2020] [Indexed: 12/24/2022] Open
Abstract
Eukaryotic DNA replication licensing is a prerequisite for, and plays a role in, regulating genome duplication that occurs exactly once per cell cycle. ORC (origin recognition complex) binds to and marks replication origins throughout the cell cycle and loads other replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs), completing replication licensing. However, how an asymmetric single-heterohexameric ORC structure loads the symmetric MCM (minichromosome maintenance) double hexamers is controversial, and importantly, it remains unknown when and how ORC proteins associate with the newly replicated origins to protect them from invasion by histones. Here, we report an essential and cell-cycle-dependent ORC "dimerization cycle" that plays three fundamental roles in the regulation of DNA replication: providing a symmetric platform to load the symmetric pre-RCs, marking and protecting the nascent sister replication origins for the next licensing, and playing a crucial role to prevent origin re-licensing within the same cell cycle.
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Affiliation(s)
- Aftab Amin
- School of Chinese Medicine and Department of Biology, Hong Kong Baptist University, Hong Kong, China; Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Rentian Wu
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Man Hei Cheung
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - John F Scott
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Ziyi Wang
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Zijing Zhou
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Changdong Liu
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Guang Zhu
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Chris Kong-Chu Wong
- School of Chinese Medicine and Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Zhiling Yu
- School of Chinese Medicine and Department of Biology, Hong Kong Baptist University, Hong Kong, China.
| | - Chun Liang
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China; The First Clinical Medicine College, Guangzhou University of Chinese Medicine, Guangzhou, China; EnKang Pharmaceuticals Limited, Guangzhou, China.
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Yang Q, Xie B, Tang H, Meng W, Jia C, Zhang X, Zhang Y, Zhang J, Li H, Fu B. Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-κB pathway. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2019; 38:263. [PMID: 31208444 PMCID: PMC6580494 DOI: 10.1186/s13046-019-1241-9] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/09/2019] [Accepted: 05/22/2019] [Indexed: 12/16/2022]
Abstract
Background Hepatocellular carcinoma (HCC) is the most common tumors in the worldwide, it develops resistance to radiotherapy during treatment, understanding the regulatory mechanisms of radioresistance generation is the urgent need for HCC therapy. Methods qRT-PCR, western blot and immunohistochemistry were used to examine MCM3 expression. MTT assay, colony formation assay, terminal deoxynucleotidyl transferase nick end labeling assay and In vivo xenograft assay were used to determine the effect of MCM3 on radioresistance. Gene set enrichment analysis, luciferase reporter assay, western blot and qRT-PCR were used to examine the effect of MCM3 on NF-κB pathway. Results We found DNA replication initiation protein Minichromosome Maintenance 3 (MCM3) was upregulated in HCC tissues and cells, patients with high MCM3 expression had poor outcome, it was an independent prognostic factor for HCC. Cells with high MCM3 expression or MCM3 overexpression increased the radioresistance determined by MTT assay, colony formation assay, TUNEL assay and orthotopic transplantation mouse model, while cells with low MCM3 expression or MCM3 knockdown reduced the radioresistance. Mechanism analysis showed MCM3 activated NF-κB pathway, characterized by increasing the nuclear translocation of p65, the expression of the downstream genes NF-κB pathway and the phosphorylation of IKK-β and IκBα. Inhibition of NF-κB in MCM3 overexpressing cells using small molecular inhibitor reduced the radioresistance, suggesting MCM3 increased radioresistance through activating NF-κB pathway. Moreover, we found MCM3 expression positively correlated with NF-κB pathway in clinic. Conclusions Our findings revealed that MCM3 promoted radioresistance through activating NF-κB pathway, strengthening the role of MCM subunits in the tumor progression and providing a new target for HCC therapy. Electronic supplementary material The online version of this article (10.1186/s13046-019-1241-9) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Qing Yang
- Department of Hepatic Surgery and Liver transplantation Center of the Third Affiliated Hospital, Organ Transplantation Institute, Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, 600# Tianhe Road, Guangzhou, 510630, China
| | - Binhui Xie
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of Gannan Medical University, Ganzhou, 341000, China
| | - Hui Tang
- Department of Hepatic Surgery and Liver transplantation Center of the Third Affiliated Hospital, Organ Transplantation Institute, Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, 600# Tianhe Road, Guangzhou, 510630, China
| | - Wei Meng
- Department of Hepatic Surgery and Liver transplantation Center of the Third Affiliated Hospital, Organ Transplantation Institute, Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, 600# Tianhe Road, Guangzhou, 510630, China
| | - Changchang Jia
- Cell-gene Therapy Translational Medicine Research Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, China
| | - Xiaomei Zhang
- Guangdong Key Laboratory of Liver Disease Research, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, China
| | - Yi Zhang
- Department of Hepatic Surgery and Liver transplantation Center of the Third Affiliated Hospital, Organ Transplantation Institute, Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, 600# Tianhe Road, Guangzhou, 510630, China
| | - Jianwen Zhang
- Department of Hepatic Surgery and Liver transplantation Center of the Third Affiliated Hospital, Organ Transplantation Institute, Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, 600# Tianhe Road, Guangzhou, 510630, China.
| | - Heping Li
- Department of Medical Oncology of the Eastern Hospital, The First Affiliated Hospital of Sun Yat-sen University, Zhongshan Er Road, Guangzhou, 510080, China.
| | - Binsheng Fu
- Department of Hepatic Surgery and Liver transplantation Center of the Third Affiliated Hospital, Organ Transplantation Institute, Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, 600# Tianhe Road, Guangzhou, 510630, China.
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Wu R, Amin A, Wang Z, Huang Y, Man-Hei Cheung M, Yu Z, Yang W, Liang C. The interaction networks of the budding yeast and human DNA replication-initiation proteins. Cell Cycle 2019; 18:723-741. [PMID: 30890025 DOI: 10.1080/15384101.2019.1586509] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G1 transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.
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Affiliation(s)
- Rentian Wu
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China.,b Guangzhou HKUST Fok Ying Tung Research Institute , Guangzhou , China
| | - Aftab Amin
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China.,b Guangzhou HKUST Fok Ying Tung Research Institute , Guangzhou , China.,c School of Chinese Medicine , Hong Kong Baptist University , Guangzhou , China
| | - Ziyi Wang
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China
| | - Yining Huang
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China
| | - Marco Man-Hei Cheung
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China.,b Guangzhou HKUST Fok Ying Tung Research Institute , Guangzhou , China
| | - Zhiling Yu
- c School of Chinese Medicine , Hong Kong Baptist University , Guangzhou , China
| | - Wei Yang
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China.,d Guangdong Lewwin Pharmaceutical Research Institute Co., Ltd , Hong Kong , China
| | - Chun Liang
- a Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience , Hong Kong University of Science and Technology , Hong Kong , China.,b Guangzhou HKUST Fok Ying Tung Research Institute , Guangzhou , China.,e ntelgen Limited , Hong Kong-Guangzhou-Foshan , China
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Tatsumi R, Ishimi Y. An MCM4 mutation detected in cancer cells affects MCM4/6/7 complex formation. J Biochem 2017; 161:259-268. [PMID: 27794528 DOI: 10.1093/jb/mvw065] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Accepted: 09/20/2016] [Indexed: 02/01/2023] Open
Abstract
An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.
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Nan YL, Hu YL, Liu ZK, Duan FF, Xu Y, Li S, Li T, Chen DF, Zeng XY. Relationships between cell cycle pathway gene polymorphisms and risk of hepatocellular carcinoma. World J Gastroenterol 2016; 22:5558-5567. [PMID: 27350734 PMCID: PMC4917616 DOI: 10.3748/wjg.v22.i24.5558] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Revised: 04/29/2016] [Accepted: 05/23/2016] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the associiations between the polymorphisms of cell cycle pathway genes and the risk of hepatocellular carcinoma (HCC).
METHODS: We enrolled 1127 cases newly diagnosed with HCC from the Tumor Hospital of Guangxi Medical University and 1200 non-tumor patients from the First Affiliated Hospital of Guangxi Medical University. General demographic characteristics, behavioral information, and hematological indices were collected by unified questionnaires. Genomic DNA was isolated from peripheral venous blood using Phenol-Chloroform. The genotyping was performed using the Sequenom MassARRAY iPLEX genotyping method. The association between genetic polymorphisms and risk of HCC was shown by P-value and the odd ratio (OR) with 95% confidence interval (CI) using the unconditional logistic regression after adjusting for age, sex, nationality, smoking, drinking, family history of HCC, and hepatitis B virus (HBV) infection. Moreover, stratified analysis was conducted on the basis of the status of HBV infection, smoking, and alcohol drinking.
RESULTS: The HCC risk was lower in patients with the MCM4 rs2305952 CC (OR = 0.22, 95%CI: 0.08-0.63, P = 0.01) and with the CHEK1 rs515255 TC, TT, TC/TT (OR = 0.73, 95%CI: 0.56-0.96, P = 0.02; OR = 0.67, 95%CI: 0.46-0.97, P = 0.04; OR = 0.72, 95%CI: 0.56-0.92, P = 0.01, respectively). Conversely, the HCC risk was higher in patients with the KAT2B rs17006625 GG (OR = 1.64, 95%CI: 1.01-2.64, P = 0.04). In addition, the risk was markedly lower for those who were carriers of MCM4 rs2305952 CC and were also HBsAg-positive and non-drinking and non-smoking (P < 0.05, respectively) and for those who were carriers of CHEK1 rs515255 TC, TT, TC/TT and were also HBsAg-negative and non-drinking (P < 0.05, respectively). Moreover, the risk was higher for those who were carriers of KAT2B rs17006625 GG and were also HBsAg-negative (P < 0.05).
CONCLUSION: Of 12 cell cycle pathway genes, MCM4, CHEK1 and KAT2B polymorphisms may be associated with the risk of HCC.
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Protein-protein interaction analysis for functional characterization of helicases. Methods 2016; 108:56-64. [PMID: 27090004 DOI: 10.1016/j.ymeth.2016.04.014] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2016] [Revised: 04/11/2016] [Accepted: 04/13/2016] [Indexed: 11/22/2022] Open
Abstract
Helicases are enzymes involved in nucleic acid metabolism, playing major roles in replication, transcription, and repair. Defining helicases oligomerization state and transient and persistent protein interactions is essential for understanding of their function. In this article we review current methods for the protein-protein interaction analysis, and discuss examples of its application to the study of helicases: Pif1 and DDX3. Proteomics methods are our main focus - affinity pull-downs and chemical cross-linking followed by mass spectrometry. We review advantages and limitations of these methods and provide general guidelines for their implementation in the functional analysis of helicases.
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Huang Y, Amin A, Qin Y, Wang Z, Jiang H, Liang L, Shi L, Liang C. A Role of hIPI3 in DNA Replication Licensing in Human Cells. PLoS One 2016; 11:e0151803. [PMID: 27057756 PMCID: PMC4825987 DOI: 10.1371/journal.pone.0151803] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Accepted: 03/06/2016] [Indexed: 01/08/2023] Open
Abstract
The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.
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Affiliation(s)
- Yining Huang
- Biomedical Research Institute, Shenzhen-PKU-HKUST Medical Center, Shenzhen, China
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
- HKUST Fok Ying Tung Research Institute, Guangzhou, China
| | - Aftab Amin
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Yan Qin
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Ziyi Wang
- Biomedical Research Institute, Shenzhen-PKU-HKUST Medical Center, Shenzhen, China
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
- HKUST Fok Ying Tung Research Institute, Guangzhou, China
| | - Huadong Jiang
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Lu Liang
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Linjing Shi
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Chun Liang
- Biomedical Research Institute, Shenzhen-PKU-HKUST Medical Center, Shenzhen, China
- Division of Life Science, Center for Cancer Research and State Key Lab for Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
- HKUST Fok Ying Tung Research Institute, Guangzhou, China
- Intelgen Ltd., Hong Kong-Guangzhou-Foshan, China
- * E-mail:
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Rizvi I, Choudhury NR, Tuteja N. Arabidopsis thaliana MCM3 single subunit of MCM2-7 complex functions as 3' to 5' DNA helicase. PROTOPLASMA 2016; 253:467-75. [PMID: 25944245 DOI: 10.1007/s00709-015-0825-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Accepted: 04/27/2015] [Indexed: 05/09/2023]
Abstract
Minichromosome maintenance 2-7 (MCM2-7) proteins are conserved eukaryotic replicative factors essential for the DNA replication at its initiation and elongation step, and act as a licensing factor. The MCM2-7 and MCM4/6/7subcomplex exhibit DNA helicase activity, and are therefore regarded as the replicative helicase. The MCM proteins have not been studied in detail in plant system. Here, we present the biochemical characterization of Arabidopsis thaliana MCM3 single subunit and show that it exhibits in vitro unwinding and ATPase activities. AtMCM3 shows a greater unwinding activity with 5' forked partial DNA duplex substrate as compared to 3' forked and non-forked substrates. ATP and magnesium ion are indispensable for its DNA helicase activity. Specifically, ATP and dATP are the preferred nucleotides for its unwinding activity. The directionality of the AtMCM3 has been determined to be in 3' to 5' direction. The oligomerization status of AtMCM3 single subunit protein indicates that it is present in different multimeric forms. The unraveling of the helicase activity of AtMCM3 will provide better insights into the plant DNA replication.
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Affiliation(s)
- Irum Rizvi
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Nirupam Roy Choudhury
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Narendra Tuteja
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
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Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2-7 Helicase to Reveal Essential Features of Structure and Function. ARCHAEA-AN INTERNATIONAL MICROBIOLOGICAL JOURNAL 2015; 2015:305497. [PMID: 26539061 PMCID: PMC4619765 DOI: 10.1155/2015/305497] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/27/2015] [Accepted: 04/05/2015] [Indexed: 11/18/2022]
Abstract
In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring.
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Ishimi Y, Irie D. G364R mutation of MCM4 detected in human skin cancer cells affects DNA helicase activity of MCM4/6/7 complex. J Biochem 2015; 157:561-9. [PMID: 25661590 DOI: 10.1093/jb/mvv015] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Accepted: 01/03/2015] [Indexed: 01/08/2023] Open
Abstract
A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30-50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.
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Affiliation(s)
- Yukio Ishimi
- College of Science, Ibaraki University, Mito, Ibaraki 351-8511, Japan
| | - Daiki Irie
- College of Science, Ibaraki University, Mito, Ibaraki 351-8511, Japan
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11
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Chen S, Qu X, Wan P, Li QW, Wang Z, Guo F, Bai L, Hu Z, Tan W, Li J. Norcantharidin inhibits pre-replicative complexes assembly of HepG2 cells. THE AMERICAN JOURNAL OF CHINESE MEDICINE 2013; 41:665-82. [PMID: 23711148 DOI: 10.1142/s0192415x13500468] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Norcantharidin (NCTD) is currently used for anticancer therapy but the exact mechanism of action remains unknown. Pre-replicative complexes (pre-RCs) are essential for cell DNA replication and highly related to malignant proliferation. Here, we examined the inhibitory effect of NCTD on pre-RC components in HepG2 cells. We showed that NCTD induced degradation of Cdc6 and Mcm2 in a dose-dependent manner. Under 100 μM NCTD concentration, about 70% of Cdc6 and 50% of Mcm2 were degraded. In addition, the nuclear translocation of Mcm6 was inhibited by NCTD. Further studies aiming at G1 synchronous cells showed that, NCTD reduced the chromatin-bound Cdc6, Mcm2 and Mcm6. Moreover, the cells were blocked from entering the S phase and accumulated at the G1 phase when released synchronously into the cell cycle. Consistently, the DNA replication was inhibited by NCTD. Finally, the combination NCTD with Cdc6 depletion lead to more severe cytotoxicity (88%) than NCTD (52%) and Cdc6 depletion (39%) alone. A synergic cytotoxicity was observed between Cdc6 depletion and NCTD. In conclusion, our results demonstrate that NCTD inhibits pre-RC assembly; subsequently blocks the G1 to S transition; and inhibits DNA replication in HepG2 cells. Pre-RCs are an intriguing target for cancer therapy, which merits further investigations for anticancer development.
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Affiliation(s)
- Sansan Chen
- Institute of Biotherapy, School of Biotechnology, Southern Medical University, Guangzhou, China
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12
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Bell SP, Kaguni JM. Helicase loading at chromosomal origins of replication. Cold Spring Harb Perspect Biol 2013; 5:cshperspect.a010124. [PMID: 23613349 DOI: 10.1101/cshperspect.a010124] [Citation(s) in RCA: 93] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.
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Affiliation(s)
- Stephen P Bell
- Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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13
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Abstract
Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2+ into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria. Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans
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Xu M, Chang YP, Chen XS. Expression, purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7. BMC BIOCHEMISTRY 2013; 14:5. [PMID: 23444842 PMCID: PMC3605359 DOI: 10.1186/1471-2091-14-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/12/2012] [Accepted: 02/13/2013] [Indexed: 11/10/2022]
Abstract
Background The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro. Results We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies. Conclusions Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.
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Affiliation(s)
- Meng Xu
- Graduate Program in Genetics, Molecular and Cell Biology, University of Southern California, Los Angeles, CA 90089, USA
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15
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Watanabe E, Ohara R, Ishimi Y. Effect of an MCM4 mutation that causes tumours in mouse on human MCM4/6/7 complex formation. J Biochem 2012; 152:191-8. [PMID: 22668557 DOI: 10.1093/jb/mvs060] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
It has been reported that a point mutation of minichromosome maintenance (MCM)4 causes mammary carcinoma, and it deregulates DNA replication to produce abnormal chromosome structures. To understand the effect of this mutation at level of MCM2-7 interaction, we examined the effect of the same mutation of human MCM4 on the complex formation with MCM6 and MCM7 in insect cells. Human MCM4/6/7 complexes containing the mutated MCM4 were formed, but the hexameric complex formation was not evident in comparison with those containing wild-type MCM4. In binary expression of MCM4 and MCM6, decreased levels of MCM6 were recovered with the mutated MCM4, compared with wild-type MCM4. These results suggest that this mutation of MCM4 perturbs proper interaction with MCM6 to affect complex formation of MCM4/6/7 that is a core structure of MCM2-7 complex. Consistent with this notion, nuclear localization and MCM complex formation of forcedly expressed MCM4 in human cells are affected by this mutation. Thus, the defect of this mutant MCM4 in interacting with MCM6 may generate a decreased level of chromatin binding of MCM2-7 complex.
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Affiliation(s)
- Emi Watanabe
- College of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 351-8511, Japan
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Interactions of the human MCM-BP protein with MCM complex components and Dbf4. PLoS One 2012; 7:e35931. [PMID: 22540012 PMCID: PMC3335088 DOI: 10.1371/journal.pone.0035931] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2011] [Accepted: 03/27/2012] [Indexed: 12/22/2022] Open
Abstract
MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.
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Huo L, Wu R, Yu Z, Zhai Y, Yang X, Chan TC, Yeung JTF, Kan J, Liang C. The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis. Cell Cycle 2012; 11:1325-39. [PMID: 22421151 DOI: 10.4161/cc.19709] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G₁ transition and for pre-RC maintenance in G₁ phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.
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Affiliation(s)
- Lin Huo
- Division of Life Science, Center for Cancer Research and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China
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Tiengwe C, Marcello L, Farr H, Gadelha C, Burchmore R, Barry JD, Bell SD, McCulloch R. Identification of ORC1/CDC6-interacting factors in Trypanosoma brucei reveals critical features of origin recognition complex architecture. PLoS One 2012; 7:e32674. [PMID: 22412905 PMCID: PMC3297607 DOI: 10.1371/journal.pone.0032674] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2011] [Accepted: 02/02/2012] [Indexed: 12/13/2022] Open
Abstract
DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.
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Affiliation(s)
- Calvin Tiengwe
- The Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
| | - Lucio Marcello
- The Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
| | - Helen Farr
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - Catarina Gadelha
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - Richard Burchmore
- Sir Henry Wellcome Functional Genomics Facility, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
| | - J. David Barry
- The Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
| | - Stephen D. Bell
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - Richard McCulloch
- The Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
- * E-mail:
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19
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Wu R, Wang J, Liang C. Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex. J Cell Sci 2012; 125:209-19. [PMID: 22250202 DOI: 10.1242/jcs.094169] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Regulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.
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Affiliation(s)
- Rentian Wu
- Division of Life Science and Center for Cancer Research, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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20
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Tran NQ, Dang HQ, Tuteja R, Tuteja N. A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase. PLANT MOLECULAR BIOLOGY 2010; 74:327-36. [PMID: 20730596 DOI: 10.1007/s11103-010-9675-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/16/2010] [Accepted: 07/27/2010] [Indexed: 05/18/2023]
Abstract
The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3'-5' DNA helicase, ATP-binding and ATPase activities. The helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.
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Affiliation(s)
- Ngoc Quang Tran
- International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
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21
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Lai F, Wu R, Wang J, Li C, Zou L, Lu Y, Liang C. Far3p domains involved in the interactions of Far proteins and pheromone-induced cell cycle arrest in budding yeast. FEMS Yeast Res 2010; 11:72-9. [PMID: 20977626 DOI: 10.1111/j.1567-1364.2010.00691.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Far3p (factor arrest), a protein that interacts with Far7-11p, is required for the pheromone-mediated cell cycle arrest in G1 phase. We used a combination of computational and experimental strategies to identify the Far3p self-association, to map the Far3p domains that interact with Far3p itself and with other Far proteins, and to reveal the importance of the two coiled-coil motifs of Far3p in the integrity and function of the Far complex. We show that Far3p self-associates through its central region and its C-terminal coiled-coil domain, that the amino acid 61-100 region of Far3p interacts with Far7p, and that the Far3p N-terminal coiled-coil domain interacts with Far9p and Far10p. Mutation of the N-terminal coiled coil disrupts the interactions of Far3p with Far9p and Far10p, and mutation of the C-terminal domain weakens the Far3p self-interaction. Although the N- and C-terminal coiled-coil mutants reserve some of the interactions with itself and some other Far proteins, both mutants are defective in the pheromone-mediated G1 arrest, indicating that both coiled-coil motifs of Far3p are essential for the integrity and the function of the Far complex.
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Affiliation(s)
- Fenju Lai
- School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Section of Biochemistry and Cell Biology, Division of Life Science, and Center for Cancer Research, Hong Kong University of Science and Technology, Hong Kong, China
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22
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Brewster AS, Chen XS. Insights into the MCM functional mechanism: lessons learned from the archaeal MCM complex. Crit Rev Biochem Mol Biol 2010; 45:243-56. [PMID: 20441442 DOI: 10.3109/10409238.2010.484836] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The helicase function of the minichromosome maintenance protein (MCM) is essential for genomic DNA replication in archaea and eukaryotes. There has been rapid progress in studies of the structure and function of MCM proteins from different organisms, leading to better understanding of the MCM helicase mechanism. Because there are a number of excellent reviews on this topic, we will use this review to summarize some of the recent progress, with particular focus on the structural aspects of MCM and their implications for helicase function. Given the hexameric and double hexameric architecture observed by X-ray crystallography and electron microscopy of MCMs from archaeal and eukaryotic cells, we summarize and discuss possible unwinding modes by either a hexameric or a double hexameric helicase. Additionally, our recent crystal structure of a full length archaeal MCM has provided structural information on an intact, multi-domain MCM protein, which includes the salient features of four unusual beta-hairpins from each monomer, and the side channels of a hexamer/double hexamer. These new structural data enable a closer examination of the structural basis of the unwinding mechanisms by MCM.
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Affiliation(s)
- Aaron S Brewster
- Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA
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23
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Minichromosome maintenance proteins 2, 3 and 7 in medulloblastoma: overexpression and involvement in regulation of cell migration and invasion. Oncogene 2010; 29:5475-89. [DOI: 10.1038/onc.2010.287] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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Numata Y, Ishihara S, Hasegawa N, Nozaki N, Ishimi Y. Interaction of human MCM2-7 proteins with TIM, TIPIN and Rb. ACTA ACUST UNITED AC 2010; 147:917-27. [DOI: 10.1093/jb/mvq028] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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Abstract
The Mcm2-7 complex serves as the eukaryotic replicative helicase, the molecular motor that both unwinds duplex DNA and powers fork progression during DNA replication. Consistent with its central role in this process, much prior work has illustrated that Mcm2-7 loading and activation are landmark events in the regulation of DNA replication. Unlike any other hexameric helicase, Mcm2-7 is composed of six unique and essential subunits. Although the unusual oligomeric nature of this complex has long hampered biochemical investigations, recent advances with both the eukaryotic as well as the simpler archaeal Mcm complexes provide mechanistic insight into their function. In contrast to better-studied homohexameric helicases, evidence suggests that the six Mcm2-7 complex ATPase active sites are functionally distinct and are likely specialized to accommodate the regulatory constraints of the eukaryotic process.
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26
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Gheorghe CP, Goyal R, Mittal A, Longo LD. Gene expression in the placenta: maternal stress and epigenetic responses. THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 2010; 54:507-23. [PMID: 19876832 PMCID: PMC2830734 DOI: 10.1387/ijdb.082770cg] [Citation(s) in RCA: 102] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Successful placental development is crucial for optimal growth, development, maturation and survival of the embryo/fetus into adulthood. Numerous epidemiologic and experimental studies have demonstrated the profound influence of intrauterine environment on life, and the diseases to which one is subject as an adult. For the most part, these invidious influences, whether maternal hypoxia, protein or caloric deficiency or excess, and others, represent types of maternal stress. In the present review, we examine certain aspects of gene expression in the placenta as a consequence of maternal stressors. To examine these issues in a controlled manner, and in a species in which the genome has been sequenced, most of these reported studies have been performed in the mouse. Although each individual maternal stress is characterized by up- or down-regulation of specific genes in the placenta, functional analysis reveals some patterns of gene expression common to the several forms of stress. Of critical importance, these genes include those involved in DNA methylation and histone modification, cell cycle regulation, and related global pathways of great relevance to epigenesis and the developmental origins of adult health and disease.
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Affiliation(s)
- Ciprian P Gheorghe
- Center for Perinatal Biology, Department of Physiology, Loma Linda University School of Medicine, Loma Linda, CA, USA
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27
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Gheorghe CP, Goyal R, Holweger JD, Longo LD. Placental gene expression responses to maternal protein restriction in the mouse. Placenta 2009; 30:411-7. [PMID: 19362366 DOI: 10.1016/j.placenta.2009.03.002] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/20/2008] [Revised: 03/04/2009] [Accepted: 03/05/2009] [Indexed: 12/15/2022]
Abstract
OBJECTIVE Maternal protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. We tested the hypothesis that, by the use of microarray technology, we could identify specific genes and cellular pathways in the developing placenta that are responsive to maternal protein deprivation, and propose a potential mechanism for observed gene expression changes. METHODS We fed pregnant FVB/NJ mice from day post-coitum 10.5 (DPC10.5) to DPC17.5, an isocaloric diet containing 50% less protein than normal chow. We used the Affymetrix Mouse 430A_2.0 array to measure gene expression changes in the placenta. We functionally annotated the regulated genes, and examined over-represented functional categories and performed pathway analysis. For selected genes, we confirmed the microarray results by use of qPCR. RESULTS We observed 244 probe sets, corresponding to 235 genes, regulated by protein restriction (p<0.001), with ninety-one genes being up-regulated, and 153 down-regulated. Up-regulated genes included those involved in the p53 pathway, apoptosis, negative regulators of cell growth, negative regulators of cell metabolism and genes related to epigenetic control. Down-regulated genes included those involved in nucleotide metabolism. CONCLUSIONS Microarray analysis has allowed us to describe the genetic response to maternal protein deprivation in the mouse placenta. We observed that negative regulators of cell growth and metabolism in conjunction with genes involved in epigenesis were up-regulated, suggesting that protein deprivation may contribute to growth restriction and long-term epigenetic changes in stressed tissues and organs. The challenge will be to understand the cellular and molecular mechanisms of these gene expression responses.
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Affiliation(s)
- C P Gheorghe
- Department of Physiology, Loma Linda University, School of Medicine, Loma Linda, CA 92350, USA
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28
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Marchini S, Mariani P, Chiorino G, Marrazzo E, Bonomi R, Fruscio R, Clivio L, Garbi A, Torri V, Cinquini M, Dell'Anna T, Apolone G, Broggini M, D'Incalci M. Analysis of gene expression in early-stage ovarian cancer. Clin Cancer Res 2009; 14:7850-60. [PMID: 19047114 DOI: 10.1158/1078-0432.ccr-08-0523] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Gene expression profile was analyzed in 68 stage I and 15 borderline ovarian cancers to determine if different clinical features of stage I ovarian cancer such as histotype, grade, and survival are related to differential gene expression. EXPERIMENTAL DESIGN Tumors were obtained directly at surgery and immediately frozen in liquid nitrogen until analysis. Glass arrays containing 16,000 genes were used in a dual-color assay labeling protocol. RESULTS Unsupervised analysis identified eight major patient partitions, one of which was statistically associated to overall survival, grading, and histotype and another with grading and histotype. Supervised analysis allowed detection of gene profiles clearly associated to histotype or to degree of differentiation. No difference was found between borderline and grade 1 tumors. As to recurrence, a subset of genes able to differentiate relapsers from nonrelapsers was identified. Among these, cyclin E and minichromosome maintenance protein 5 were found particularly relevant, as their expression was inversely correlated to progression-free survival (P = 0.00033 and 0.017, respectively). CONCLUSIONS Specific molecular signatures define different histotypes and prognosis of stage I ovarian cancer. Mucinous and clear cells histotypes can be distinguished from the others regardless of tumor grade. Cyclin E and minichromosome maintenance protein 5, whose expression was found previously to be related to a bad prognosis of advanced ovarian cancer, appear to be potential prognostic markers in stage I ovarian cancer too, independent of other pathologic and clinical variables.
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Affiliation(s)
- Sergio Marchini
- Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
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Zhang GQ, Wang H, Liu WT, Dong H, Fong WF, Tang LM, Xiong YH, Yu ZL, Ko KM. Long-Term Treatment with a Chinese Herbal Formula, Sheng-Mai-San, Improves Cardiac Contractile Function in Aged Rats: The Role of Ca2+Homeostasis. Rejuvenation Res 2008; 11:991-1000. [DOI: 10.1089/rej.2008.0771] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Guang-Qin Zhang
- Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Hui Wang
- School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
| | - Wen-Tao Liu
- Department of Pharmacology, Nanjing Medical University, Nanjing, China
| | - Hang Dong
- School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
| | - Wang-Fun Fong
- School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
| | - Li-Min Tang
- Laboratory for Quality Control, Jiangxi Jurentang Pharmaceutical Co. Ltd., Nanchang, China
| | - Yun-Hua Xiong
- Laboratory for Quality Control, Jiangxi Jurentang Pharmaceutical Co. Ltd., Nanchang, China
| | - Zhi-Ling Yu
- School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
| | - Kam-Ming Ko
- Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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Kanter DM, Bruck I, Kaplan DL. Mcm subunits can assemble into two different active unwinding complexes. J Biol Chem 2008; 283:31172-82. [PMID: 18801730 DOI: 10.1074/jbc.m804686200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.
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Affiliation(s)
- Diane M Kanter
- Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA
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Subunit organization of Mcm2-7 and the unequal role of active sites in ATP hydrolysis and viability. Mol Cell Biol 2008; 28:5865-73. [PMID: 18662997 DOI: 10.1128/mcb.00161-08] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA(+) ATPase and the putative eukaryotic replicative helicase. Unlike a typical homohexameric helicase, Mcm2-7 contains six distinct, essential, and evolutionarily conserved subunits. Precedence to other AAA(+) proteins suggests that Mcm ATPase active sites are formed combinatorially, with Walker A and B motifs contributed by one subunit and a catalytically essential arginine (arginine finger) contributed by the adjacent subunit. To test this prediction, we used copurification experiments to identify five distinct and stable Mcm dimer combinations as potential active sites; these subunit associations predict the architecture of the Mcm2-7 complex. Through the use of mutant subunits, we establish that at least three sites are active for ATP hydrolysis and have a canonical AAA(+) configuration. In isolation, these five active-site dimers have a wide range of ATPase activities. Using Walker B and arginine finger mutations in defined Mcm subunits, we demonstrate that these sites similarly make differential contributions toward viability and ATP hydrolysis within the intact hexamer. Our conclusions predict a structural discontinuity between Mcm2 and Mcm5 and demonstrate that in contrast to other hexameric helicases, the six Mcm2-7 active sites are functionally distinct.
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Abstract
Eukaryotic DNA replication is regulated to ensure all chromosomes replicate once and only once per cell cycle. Replication begins at many origins scattered along each chromosome. Except for budding yeast, origins are not defined DNA sequences and probably are inherited by epigenetic mechanisms. Initiation at origins occurs throughout the S phase according to a temporal program that is important in regulating gene expression during development. Most replication proteins are conserved in evolution in eukaryotes and archaea, but not in bacteria. However, the mechanism of initiation is conserved and consists of origin recognition, assembly of prereplication (pre-RC) initiative complexes, helicase activation, and replisome loading. Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can only occur in G1 phase, whereas helicase activation and loading can only occur in S phase. Checkpoint regulation maintains high fidelity by stabilizing replication forks and preventing cell cycle progression during replication stress or damage.
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Affiliation(s)
- R A Sclafani
- Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.
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Komamura-Kohno Y, Tanaka R, Omori A, Kohno T, Ishimi Y. Biochemical characterization of fragmented human MCM2. FEBS J 2008; 275:727-38. [PMID: 18190532 DOI: 10.1111/j.1742-4658.2007.06239.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The molecular dissection of human MCM2, a constituent of MCM2-7 licensing factor complex, was performed to identify the region responsible for its biochemical activities. Partial digestion with trypsin dissected the MCM2 protein into a central region (148-676) containing ATPase motifs and a C-terminal region (677-895). These two fragments, along with three other fragments (148-441, 442-676 and 442-895), were produced using the wheat germ cell-free system and were examined for their ability to inhibit MCM4/6/7 helicase activity. Two fragments (442-895 and 677-895) containing the C-terminus were partly inhibitory to the activity. Further dissection revealed that one fragment (713-895) has strong inhibitory activity. The inhibitory activity of the smaller fragments derived from the C-terminal region correlated with their ability to inhibit SV40 T antigen helicase activity and also with their ability to bind to ssDNA, which has been shown by gel mobility shift analysis. These results strongly suggest that the MCM2 fragments derived from the C-terminal region inhibit DNA helicase activity through their ability to bind to ssDNA. In contrast, two fragments (148-441 and 442-676) from the central region were mainly responsible for the interaction between MCM2 and MCM4, and this was revealed by a pulldown analysis using MCM4 protein beads. Finally, only complete MCM2, not the smaller fragments, could disassemble the MCM4/6/7 hexamer into the MCM2/4/6/7 tetramer.
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Affiliation(s)
- Yuki Komamura-Kohno
- Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan
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Maeng YH, Kang HW, Huh JS. The Expression and Clinical Significance of the Minichromosome Maintenance (MCM) 7 Proliferation Markers in Urothelial Carcinomas of the Bladder. Korean J Urol 2008. [DOI: 10.4111/kju.2008.49.1.12] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Affiliation(s)
- Young-Hee Maeng
- Department of Patholgy, School of Medicine, Cheju University, Jeju, Korea
| | - Hyun Wook Kang
- Department of Forensic Medicine, School of Medicine, Cheju University, Jeju, Korea
| | - Jung-Sik Huh
- Department of Urology, School of Medicine, Cheju University, Jeju, Korea
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Minichromosome maintenance proteins 2 and 5 in non-benign epithelial ovarian tumours: relationship with cell cycle regulators and prognostic implications. Br J Cancer 2007; 97:1124-34. [PMID: 17940502 PMCID: PMC2360432 DOI: 10.1038/sj.bjc.6603992] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Minichromosome maintenance proteins (MCM) have recently emerged as novel proliferation markers with prognostic implications in several tumour types. This is the first study investigating MCM-2 and MCM-5 immunohistochemical expression in a series of ovarian adenocarcinomas and low malignant potential (LMP) tumours aiming to determine possible associations with clinicopathological parameters, the conventional proliferation index Ki-67, cell cycle regulators (p53, p27(Kip1), p21(WAF1) and pRb) and patients' outcome. Immunohistochemistry was applied in a series of 43 cases of ovarian LMP tumours and 85 cases of adenocarcinomas. Survival analysis was restricted to adenocarcinomas. The median MCM-2 and MCM-5 labelling indices (LIs) were significantly higher in adenocarcinomas compared to LMP tumours (P<0.0001 for both associations). In adenocarcinomas, the levels of MCM-2 and MCM-5 increased significantly with advancing tumour stage (P=0.0052 and P=0.0180, respectively), whereas both MCM-2 and MCM-5 increased significantly with increasing tumour grade (P=0.0002 and P=0.0006, respectively) and the presence of bulky residual disease (P<0.0001 in both relationships). A strong positive correlation was established between MCM-2 or MCM-5 expression level and Ki-67 LI (P<0.0001) as well as p53 protein (P=0.0038 and P=0.0500, respectively). Moreover, MCM-2 LI was inversely correlated with p27(Kip-1) LI (P=0.0068). Finally, both MCM-2 and MCM-5 were associated significantly with adverse patients' outcome in both univariate (> or =20 vs >20%, P=0.0011 and > or =25 vs <25%, P=0.0100, respectively) and multivariate (P=0.0001 and 0.0090, respectively) analysis. An adequately powered independent group of 45 patients was used in order to validate our results in univariate survival analysis. In this group, MCM-2 and MCM-5 expression retained their prognostic significance (P<0.0001 in both relationships). In conclusion, MCM-2 and MCM-5 proteins appear to be promising as prognostic markers in patients with ovarian adenocarcinomas.
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36
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Rothenberg E, Trakselis MA, Bell SD, Ha T. MCM forked substrate specificity involves dynamic interaction with the 5'-tail. J Biol Chem 2007; 282:34229-34. [PMID: 17884823 DOI: 10.1074/jbc.m706300200] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The archaeal minichromosome maintenance protein MCM forms a homohexameric complex that functions as the DNA replicative helicase and serves as a model system for its eukaryotic counterpart. Here, we applied single molecule fluorescence resonance energy transfer methods to probe the substrate specificity and binding mechanism of MCM from the hyperthermophilic Archaea Sulfolobus solfataricus on various DNA substrates. S. solfataricus MCM displays a binding preference for forked substrates relative to partial or full duplex substrates. Moreover, the nature of MCM binding to Y-shaped substrates is distinct in that MCM loads on the 3'-tail while interacting with the 5'-tail likely via the MCM surface. These results provide the first elucidation of a dynamic nature of interaction between a ring-shaped helicase interacting with an opposing single-stranded DNA tail. This interaction contributes to substrate selectivity and increases the stability of the forked DNA-MCM complex, with possible implications for the MCM unwinding mechanism.
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Affiliation(s)
- Eli Rothenberg
- Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, IL 61801, USA
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Sakwe AM, Nguyen T, Athanasopoulos V, Shire K, Frappier L. Identification and characterization of a novel component of the human minichromosome maintenance complex. Mol Cell Biol 2007; 27:3044-55. [PMID: 17296731 PMCID: PMC1899943 DOI: 10.1128/mcb.02384-06] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G(1) but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.
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Affiliation(s)
- Amos M Sakwe
- Department of Medical Genetics, University of Toronto, Kings College Circle, Toronto, Ontario, Canada
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Patterson S, Robert C, Whittle C, Chakrabarti R, Doerig C, Chakrabarti D. Pre-replication complex organization in the atypical DNA replication cycle of Plasmodium falciparum: Characterization of the mini-chromosome maintenance (MCM) complex formation. Mol Biochem Parasitol 2006; 145:50-9. [PMID: 16257456 DOI: 10.1016/j.molbiopara.2005.09.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2005] [Revised: 09/09/2005] [Accepted: 09/15/2005] [Indexed: 10/25/2022]
Abstract
The overall organization of cell division in Plasmodium is unique compared to that observed in model organisms because DNA replicates more than once per cell cycle at several points of its life cycle. The sequencing of the Plasmodium genome has also revealed the apparent absence of many key components (e.g. Cdt1, DDK and Cdc45) of the eukaryotic cell cycle machinery that are responsible for the formation of the pre-replication complex (pre-RC). We have characterized the Plasmodium falciparum minichromosome maintenance complex (MCM) that plays a key role in the transition of pre-RC to the RC. Similar to other eukaryotes, the Plasmodium genome encodes six MCM subunits. Here, we show that expression levels of at least three of the PfMCM subunits, the homologues of MCM2, MCM6 and MCM7, change during the intraerythrocytic development cycle, peaking in schizont and decreasing in the ring and trophozoite stages. PfMCM2, 6 and 7 subunits interact with each other to form a developmentally regulated complex: these interactions are detectable in rings and schizonts, but not in trophozoites. PfMCM2, 6 and 7 subunits are localized in both cytosolic and nucleosolic fractions during all intraerythrocytic stages of P. falciparum development, with increased nuclear localization in schizonts. Only PfMCM6 is associated with the chromatin fraction at all stages of growth. No phosphorylation of PfMCM2, 6 and 7 was detected, but two as yet unidentified threonine-phosphosphorylated proteins were present in the complex, whose pattern of phosphorylation varied during parasite development.
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Affiliation(s)
- Shelley Patterson
- Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32826, USA
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Korkolopoulou P, Givalos N, Saetta A, Goudopoulou A, Gakiopoulou H, Thymara I, Thomas-Tsagli E, Patsouris E. Minichromosome maintenance proteins 2 and 5 expression in muscle-invasive urothelial cancer: a multivariate survival study including proliferation markers and cell cycle regulators. Hum Pathol 2005; 36:899-907. [PMID: 16112007 DOI: 10.1016/j.humpath.2005.06.008] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2005] [Accepted: 06/14/2005] [Indexed: 01/10/2023]
Abstract
Evaluation of cell cycle regulators has gained special interest in the effort to increase the amount of prognostic information in malignant tumors. Minichromosome maintenance proteins (MCMs) drive the formation of prereplicative complexes, which is the first key event during G1 phase. Therefore, altered MCM expression may be a hallmark of cell cycle deregulation, which is supposed to be the most essential mechanism in the development and progression of bladder cancer. Our aim was to investigate the value of MCMs as proliferation markers and prognostic indicators in detrusor muscle-invasive urothelial bladder carcinomas. We analyzed immunohistochemically the expression of MCM-2 and MCM-5 in 65 patients with detrusor muscle-invasive urothelial bladder carcinomas in relation with clinicopathologic parameters, patients' overall and disease-free survival, and the expression of the conventional proliferation index Ki-67 and other cell cycle modulators (p53, pRb, p21(WAF1), and p27(Kip1)). The levels of MCM-2 and MCM-5 were significantly higher in high-grade (P < .0001), advanced-stage (P = .001), and nonpapillary tumors (P < .0001). The expression of MCM-2 and MCM-5 significantly associated with the conventional proliferation index Ki-67 (P = .0001 for each protein). The expression of MCM-2 or MCM-5 positively correlated with p53 labeling index (P = .014 and P = .009, respectively). Also, median p21(WAF1) labeling index was higher in MCM-5 high expressors (P = .028). Finally, both MCM-2 and MCM-5 associated significantly with adverse patients' outcome in both univariate (P = .0072 and P = .0074, respectively) and multivariate (P = .0001) analysis. In conclusion, MCM-2 and MCM-5 appear to be reliable proliferation indexes and useful prognostic markers in patients with muscle-invasive urothelial bladder carcinomas.
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