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Sandeep Saxena - One of the best experts on this subject based on the ideXlab platform.
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crl4 ddb1 vprbp ubiquitin ligase mediates the stress triggered proteolysis of MCM10
Nucleic Acids Research, 2012Co-Authors: Manpreet Kaur, Ananya Kar, Aparna Sharma, Md Muntaz Khan, Sandeep SaxenaAbstract:When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of MCM10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that MCM10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered MCM10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates MCM10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates MCM10 in vitro, proving that MCM10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets MCM10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, MCM10.
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CRL4–DDB1–VPRBP ubiquitin ligase mediates the stress triggered proteolysis of MCM10
Nucleic acids research, 2012Co-Authors: Manpreet Kaur, Muntaz Khan, Ananya Kar, Aparna Sharma, Sandeep SaxenaAbstract:When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of MCM10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that MCM10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered MCM10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates MCM10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates MCM10 in vitro, proving that MCM10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets MCM10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, MCM10.
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MCM10 proteolysis initiates before the onset of M-phase.
BMC cell biology, 2010Co-Authors: Manpreet Kaur, Muntaz Khan, Ananya Kar, Aparna Sharma, Sandeep SaxenaAbstract:Background MCM10 protein is essential for initiation and elongation phases of replication. Human cells proteolyze MCM10 during mitosis, presumably to ensure a single round of replication. It has been proposed that anaphase promoting complex ubiquitinates MCM10 in late M and early G1 phases.
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Ultraviolet Radiation Stress Triggers the Down-regulation of Essential Replication Factor MCM10
The Journal of biological chemistry, 2010Co-Authors: Aparna Sharma, Manpreet Kaur, Ananya Kar, Sourabh M. Ranade, Sandeep SaxenaAbstract:We report that upon UV radiation insult, mammalian cells specifically down-regulate MCM10, a protein essential for the initiation and elongation phases of DNA replication. The levels of a majority of replication factors remain unaffected under this condition, implying that MCM10 is a key node in the regulation of the replication machinery. High doses of ionizing gamma radiation and exposure to a combination of DNA-damaging chemicals do not decrease MCM10 protein levels, demonstrating that MCM10 down-regulation is triggered only by UV-specific damage. The decrease of MCM10 protein levels is not caused by transcriptional inhibition or cleavage by apoptotic enzymes, but results from degradation by the 26 S proteasome. UV-triggered degradation of MCM10 requires its linker or C-terminal domain. In addition, MCM10 down-regulation is not limited to cells from a particular lineage. Therefore, our study reveals a mechanism by which mammalian cells effectively inhibit the replication machinery during stress to prevent it from drifting toward a catastrophic path of genomic instability.
Anja Katrin Bielinsky - One of the best experts on this subject based on the ideXlab platform.
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4547 Understanding the molecular mechanism of natural killer cell deficiency to improve natural killer cell in vitro differentiation for therapeutics
Journal of Clinical and Translational Science, 2020Co-Authors: Megan Schmit, Ryan M Baxley, Emily M. Mace, Jordan S. Orange, Jeffery Miller, Anja Katrin BielinskyAbstract:OBJECTIVES/GOALS: Natural killer (NK) cells are a potential cancer therapeutic but expanding NK cells efficiently in vitro is difficult. Natural killer cell deficiency (NKD), a primary immune deficiency affecting only NK cells, is caused by defects in several DNA replication proteins. By studying NKD we will achieve better NK cell in vitro differentiation. METHODS/STUDY POPULATION: One patient with NKD has a compound heterozygous mutation in the essential DNA replication protein MCM10. We hypothesize that in individuals with NKD, dramatic telomere erosion from abnormal DNA replication leads to premature senescence and the loss of NK cells. To test our hypothesis, we will knockout one allele of MCM10 or over express MCM10 in NK cells isolated from blood. We will then monitor telomere length, expansion and cytotoxic activity of these NK cells. To understand the role of MCM10 in early stages of NK cell development we will deplete MCM10 in induced pluripotent stem cells and differentiate these cells into NK cells. During this differentiation we will monitor progression through NK cell developmental stages as well as telomere length and senescence markers. RESULTS/ANTICIPATED RESULTS: Telomeres insulate chromosomes and induce permanent growth arrest (senescence) when they are critically short. We have demonstrated that depletion of a DNA replication protein causes telomere erosion and increases senescence markers. NK cells have shorter telomeres and lower telomerase expression than other immune cells. We predict, this relatively poor telomere maintenance sensitizes NK cells to telomere loss upon depletion of replication proteins. During in vitro differentiation, we expect NK cell precursors to undergo premature senescence secondary to telomere shortening. Furthermore, we expect supplementation of DNA replication proteins will enhance NK cell expansion and maturation. DISCUSSION/SIGNIFICANCE OF IMPACT: NKD patients have provided the scientific community with clues as to what proteins NK cells rely on for their development. This project aims not only to understand why these proteins are critical, but to harness that information for cellular anti-cancer therapeutics.
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MCM10: A Dynamic Scaffold at Eukaryotic Replication Forks.
Genes, 2017Co-Authors: Ryan M Baxley, Anja Katrin BielinskyAbstract:To complete the duplication of large genomes efficiently, mechanisms have evolved that coordinate DNA unwinding with DNA synthesis and provide quality control measures prior to cell division. Minichromosome maintenance protein 10 (MCM10) is a conserved component of the eukaryotic replisome that contributes to this process in multiple ways. MCM10 promotes the initiation of DNA replication through direct interactions with the cell division cycle 45 (Cdc45)-minichromosome maintenance complex proteins 2-7 (Mcm2-7)-go-ichi-ni-san GINS complex proteins, as well as single- and double-stranded DNA. After origin firing, MCM10 controls replication fork stability to support elongation, primarily facilitating Okazaki fragment synthesis through recruitment of DNA polymerase-α and proliferating cell nuclear antigen. Based on its multivalent properties, MCM10 serves as an essential scaffold to promote DNA replication and guard against replication stress. Under pathological conditions, MCM10 is often dysregulated. Genetic amplification and/or overexpression of MCM10 are common in cancer, and can serve as a strong prognostic marker of poor survival. These findings are compatible with a heightened requirement for MCM10 in transformed cells to overcome limitations for DNA replication dictated by altered cell cycle control. In this review, we highlight advances in our understanding of when, where and how MCM10 functions within the replisome to protect against barriers that cause incomplete replication.
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Mapping ubiquitination sites of S. cerevisiae MCM10
Biochemistry and biophysics reports, 2016Co-Authors: Tianji Zhang, Brandy L. Fultz, Sapna Das-bradoo, Anja Katrin BielinskyAbstract:Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, MCM10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between MCM10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of MCM10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae MCM10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets MCM10 and that ubiquitination has a role in suppressing replication stress.
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Slx5/Slx8 Promotes Replication Stress Tolerance by Facilitating Mitotic Progression
Cell reports, 2016Co-Authors: Yee Mon Thu, Tianji Zhang, Jordan R. Becker, Hai Dang Nguyen, Susan K. Van Riper, Leeann Higgins, Todd W. Markowski, Timothy J. Griffin, Anja Katrin BielinskyAbstract:Loss of minichromosome maintenance protein 10 (MCM10) causes replication stress. We uncovered that S. cerevisiae MCM10-1 mutants rely on the E3 SUMO ligase Mms21 and the SUMO-targeted ubiquitin ligase complex Slx5/8 for survival. Using quantitative mass spectrometry, we identified changes in the SUMO proteome of MCM10-1 mutants and revealed candidates regulated by Slx5/8. Such candidates included subunits of the chromosome passenger complex (CPC), Bir1 and Sli15, known to facilitate spindle assembly checkpoint (SAC) activation. We show here that Slx5 counteracts SAC activation in MCM10-1 mutants under conditions of moderate replication stress. This coincides with the proteasomal degradation of sumoylated Bir1. Importantly, Slx5-dependent mitotic relief was triggered not only by MCM10 deficiency but also by treatment with low doses of the alkylating drug methyl methanesulfonate. Based on these findings, we propose a model in which Slx5/8 allows for passage through mitosis when replication stress is tolerable.
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The Role of MCM10 in Replication Initiation
The Initiation of DNA Replication in Eukaryotes, 2016Co-Authors: Ryan M Baxley, Yee Mon Thu, Anja Katrin BielinskyAbstract:Minichromosome maintenance protein 10 (MCM10) is a conserved component of the eukaryotic DNA replication machinery. MCM10 promotes the initiation of replication by facilitating DNA unwinding and origin firing. Although the molecular details of this action remain unclear, current data support a scaffolding role for MCM10 via interactions with DNA and other protein partners. MCM10 binds both single- and double-stranded DNA, as well as components of the CMG helicase complex, DNA polymerase-α, and Ctf4. Upon initiation, MCM10 becomes part of the replisome, primarily mediating the initiation of Okazaki fragment synthesis, which involves DNA polymerase-α/primase and the replication clamp PCNA. MCM10 likely contributes to the recruitment of both of these factors. Emerging concepts predict that steady-state levels of MCM10 are tightly controlled to balance origin firing and fork progression. Investigations into the cellular requirements for MCM10 have also revealed a key role in maintaining genome stability. Accordingly, it is not surprising that genetic alterations of MCM10 are associated with cancer. Loss of MCM10 function is a possible source of DNA damage, whereas overexpression of MCM10 might serve to facilitate rapid DNA synthesis and proliferation. In this chapter, we provide a comprehensive review of the current literature describing MCM10’s role in replication initiation. Additionally, we consider how contributions to elongation and other potential functions may affect chromosomal integrity.
Manpreet Kaur - One of the best experts on this subject based on the ideXlab platform.
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crl4 ddb1 vprbp ubiquitin ligase mediates the stress triggered proteolysis of MCM10
Nucleic Acids Research, 2012Co-Authors: Manpreet Kaur, Ananya Kar, Aparna Sharma, Md Muntaz Khan, Sandeep SaxenaAbstract:When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of MCM10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that MCM10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered MCM10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates MCM10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates MCM10 in vitro, proving that MCM10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets MCM10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, MCM10.
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CRL4–DDB1–VPRBP ubiquitin ligase mediates the stress triggered proteolysis of MCM10
Nucleic acids research, 2012Co-Authors: Manpreet Kaur, Muntaz Khan, Ananya Kar, Aparna Sharma, Sandeep SaxenaAbstract:When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of MCM10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that MCM10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered MCM10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates MCM10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates MCM10 in vitro, proving that MCM10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets MCM10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, MCM10.
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MCM10 proteolysis initiates before the onset of M-phase.
BMC cell biology, 2010Co-Authors: Manpreet Kaur, Muntaz Khan, Ananya Kar, Aparna Sharma, Sandeep SaxenaAbstract:Background MCM10 protein is essential for initiation and elongation phases of replication. Human cells proteolyze MCM10 during mitosis, presumably to ensure a single round of replication. It has been proposed that anaphase promoting complex ubiquitinates MCM10 in late M and early G1 phases.
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Ultraviolet Radiation Stress Triggers the Down-regulation of Essential Replication Factor MCM10
The Journal of biological chemistry, 2010Co-Authors: Aparna Sharma, Manpreet Kaur, Ananya Kar, Sourabh M. Ranade, Sandeep SaxenaAbstract:We report that upon UV radiation insult, mammalian cells specifically down-regulate MCM10, a protein essential for the initiation and elongation phases of DNA replication. The levels of a majority of replication factors remain unaffected under this condition, implying that MCM10 is a key node in the regulation of the replication machinery. High doses of ionizing gamma radiation and exposure to a combination of DNA-damaging chemicals do not decrease MCM10 protein levels, demonstrating that MCM10 down-regulation is triggered only by UV-specific damage. The decrease of MCM10 protein levels is not caused by transcriptional inhibition or cleavage by apoptotic enzymes, but results from degradation by the 26 S proteasome. UV-triggered degradation of MCM10 requires its linker or C-terminal domain. In addition, MCM10 down-regulation is not limited to cells from a particular lineage. Therefore, our study reveals a mechanism by which mammalian cells effectively inhibit the replication machinery during stress to prevent it from drifting toward a catastrophic path of genomic instability.
Bik Kwoon Tye - One of the best experts on this subject based on the ideXlab platform.
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Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase
Molecular and cellular biology, 2009Co-Authors: Chanmi Lee, Ivan Liachko, Roxane Bouten, Zvi Kelman, Bik Kwoon TyeAbstract:Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase alpha in the absence of their presumed linker, MCM10. MCM10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polalpha. Dominant mcm2 suppressors of MCM10 mutants restore viability by restoring the stability of Polalpha without restoring the stability of MCM10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the MCM10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that MCM10 plays two important roles as a linker of the MCM helicase and Polalpha at the elongating replication fork--first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.
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MCM10 mediates the interaction between DNA replication and silencing machineries.
Genetics, 2008Co-Authors: Ivan Liachko, Bik Kwoon TyeAbstract:The connection between DNA replication and heterochromatic silencing in yeast has been a topic of investigation for >20 years. While early studies showed that silencing requires passage through S phase and implicated several DNA replication factors in silencing, later works showed that silent chromatin could form without DNA replication. In this study we show that members of the replicative helicase (Mcm3 and Mcm7) play a role in silencing and physically interact with the essential silencing factor, Sir2, even in the absence of DNA replication. Another replication factor, MCM10, mediates the interaction between these replication and silencing proteins via a short C-terminal domain. Mutations in this region of MCM10 disrupt the interaction between Sir2 and several of the Mcm2–7 proteins. While such mutations caused silencing defects, they did not cause DNA replication defects or affect the association of Sir2 with chromatin. Our findings suggest that MCM10 is required for the coupling of the replication and silencing machineries to silence chromatin in a context outside of DNA replication beyond the recruitment and spreading of Sir2 on chromatin.
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MCM10 Is Required for the Maintenance of Transcriptional Silencing in Saccharomyces cerevisiae
Genetics, 2005Co-Authors: Ivan Liachko, Bik Kwoon TyeAbstract:MCM10 is an essential protein that participates in both the initiation and the elongation of DNA replication. In this study we demonstrate a role for MCM10 in the maintenance of heterochromatic silencing at telomeres and HM loci of budding yeast. Two MCM10 mutants drastically reduce silencing of both URA3 and ADE2 reporter genes integrated into these silent loci. When exposed to α-factor, MCM10 mutant cells display a “shmoo-cluster” phenotype associated with a defect in the maintenance of silencing. In addition, when combined with a defect in the establishment of silent chromatin, MCM10 mutants demonstrate a synergistic defect in HML silencing. Consistent with a direct silencing function, MCM10p shows a two-hybrid interaction with Sir2p and Sir3p that is destroyed by the MCM10-1 mutation and dependent on the C-terminal 108 amino acids. Tethering GBD-MCM10 to a defective HMR-E silencer is not sufficient to restore silencing. Furthermore, mutations in MCM10 inhibit the ability of GBD-SIR3 to restore silencing when tethered to a defective HMR-E . Suppressor mutations in MCM2 , which suppress the temperature sensitivity of MCM10-1 , fail to overcome the MCM10-1 silencing defect, suggesting that MCM10 9s role in transcriptional silencing may be separate from its essential functions in DNA replication.
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MCM10 and Cdc45 Cooperate in Origin Activation in Saccharomyces cerevisiae
Journal of molecular biology, 2004Co-Authors: Sara L. Sawyer, Irene H. Cheng, Weihang Chai, Bik Kwoon TyeAbstract:MCM10 has recently been found to play a crucial role in multiple steps of the DNA replication initiation process in eukaryotes. Here, we have examined the role of MCM10 in assembling initiation factors at a well-characterized yeast replication origin, ARS1. We find that the pre-replication complex (pre-RC) components Cdc6 and Mcm7 associate with ARS1 in the MCM10-1 mutant, suggesting that establishment of the pre-RC is not compromised in this mutant. Association of Cdc45 with ARS1 is reduced in the MCM10-1 mutant, suggesting that MCM10 is involved in recruiting Cdc45 to the pre-RC. We find that overexpression of either MCM10-1 or Cdc45 suppresses the growth defect of MCM10-1, and that a physical interaction between Cdc45 and MCM10 is disrupted in the MCM10-1 mutant. Our results show that interaction between the MCM10 and Cdc45 proteins facilitates the recruitment of Cdc45 onto the ARS1 origin.
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budding yeast MCM10 dna43 mutant requires a novel repair pathway for viability
Genes to Cells, 2003Co-Authors: Yoshio Araki, Yasuo Kawasaki, Hiroyuki Sasanuma, Bik Kwoon Tye, Akio SuginoAbstract:Background:MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. MCM10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC. Two-dimensional gel electrophoresis analysis showed that MCM10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Polδ and Polɛ, suggesting that it may play a role in elongation replication. Results: We show that the MCM10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with MCM10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with MCM10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6. Conclusions: The viability of the MCM10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that MCM10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.
Brandt F. Eichman - One of the best experts on this subject based on the ideXlab platform.
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The anti-parasitic agent suramin and several of its analogues are inhibitors of the DNA binding protein MCM10
Open biology, 2019Co-Authors: Carolyn N. Paulson, Ryan M Baxley, Brandt F. Eichman, Walter J. Chazin, Kristen John, Fredy Kurniawan, Kayo Orellana, Rawle Francis, Alexandra Sobeck, Hideki AiharaAbstract:Minichromosome maintenance protein 10 (MCM10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit MCM10 from binding to DNA. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human MCM10 internal domain and full-length Xenopus MCM10, including analogues that are selective for MCM10 over human RPA. Binding of suramin analogues to MCM10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human MCM10 binding affinity (FP Ki 170 nM, SPR KD 460 nM) and cell activity (IC50 38 µM). Suramin and its analogues are the first identified inhibitors of MCM10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.
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MCM10 self-association is mediated by an N-terminal coiled-coil domain.
PloS one, 2013Co-Authors: Ajeetha Josephrajan, Anja Katrin Bielinsky, Suraj Adhikary, Timothy M. Bowles, Brandt F. EichmanAbstract:Minichromosome maintenance protein 10 (MCM10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. MCM10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis MCM10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 A resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric MCM10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that MCM10 likely forms dimers or trimers to promote its diverse functions during DNA replication.
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Structural biology of replication initiation factor MCM10.
Sub-cellular biochemistry, 2012Co-Authors: Melissa E. Stauffer, Brandt F. EichmanAbstract:Minichromosome maintenance protein 10 (MCM10) is a non-enzymatic replication factor required for proper assembly of the eukaryotic replication fork. MCM10 interacts with single-stranded and double-stranded DNA, DNA polymerase α and Mcm2-7, and is important for activation of the pre-replicative complex and recruitment of subsequent proteins to the origin at the onset of S-phase. In addition, MCM10 has recently been implicated in coordination of helicase and polymerase activities during replication fork progression. The nature of MCM10’s involvement in these activities, whether direct or indirect, remains unknown. However, recent biochemical and structural characterization of MCM10 from multiple organisms has provided insights into how MCM10 utilizes a modular architecture to act as a replisome scaffold, which helps to define possible roles in origin DNA melting, Pol α recruitment and coordination of enzymatic activities during elongation.
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Solution NMR structure of the C-terminal DNA binding domain of MCM10 reveals a conserved MCM motif
The Journal of biological chemistry, 2010Co-Authors: Patrick D. Robertson, Benjamin Chagot, Walter J. Chazin, Brandt F. EichmanAbstract:The eukaryotic DNA replication protein MCM10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) MCM10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase α is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMCM10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to MCM10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate MCM10. Our results provide insight in the action of MCM10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.
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Physical Interactions between MCM10, DNA, and DNA Polymerase α
The Journal of biological chemistry, 2009Co-Authors: Eric M. Warren, Ellen Fanning, Walter J. Chazin, Hao Huang, Brandt F. EichmanAbstract:MCM10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, MCM10 is required for the association of several replication proteins, including DNA polymerase alpha (pol alpha), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of MCM10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol alpha. However, the mechanism by which MCM10 interacts with pol alpha on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between MCM10-ID, ssDNA, and p180. The crystal structure of an MCM10-ID*ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of MCM10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal MCM10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286-310). These findings, together with data for DNA and p180 binding to an MCM10 construct that contains both the ID and CTD, provide the first mechanistic insight into how MCM10 might use a handoff mechanism to load and stabilize pol alpha within the replication fork.