MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication
Debra L. EvansHaoxing ZhangHyoungjun HamHuadong PeiSeungBaek LeeJung-Jin KimDaniel D. BilladeauZhenkun Lou
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Abstract:
The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4Cdt2) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.Keywords:
Origin recognition complex
Minichromosome maintenance
Pre-replication complex
DNA re-replication
Licensing factor
S phase
Replication factor C
The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.
Origin recognition complex
Minichromosome maintenance
Replication factor C
Licensing factor
S phase
Pre-replication complex
DNA re-replication
Replication protein A
Cyclin A
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Minichromosome maintenance
Origin recognition complex
Pre-replication complex
DNA re-replication
Licensing factor
S phase
Replication factor C
Chromothripsis
Replication
Semiconservative replication
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Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information. The molecular mechanism that guarantees that many origins of replication fire only once per cell–cycle has been the area of intense research. The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre–replicative complex (pre–RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10–dependent transcript (Cdt1) and mini–chromosome maintenance (MCM) proteins. The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once–per–cell–cycle and its binding to the chromatin marks the final step of pre–RC formation, a process referred to as ‘replication licensing’. We present data demonstrating how the MCM proteins associate with the chromatin during the G1 phase, probably defining pre–RCs and then anticipate replication fork movement in a precisely coordinated manner during the S phase of the cell cycle. The process of DNA replication must also be carefully coordinated with other cell–cycle processes including mitosis and cytokinesis. Some of the proteins that control initiation of DNA replication are likely to interact with the pathways that control these important cell–cycle transitions. Herein, we discuss the participation of human ORC proteins in other vital functions, in addition to their bona fide roles in replication.
Pre-replication complex
Origin recognition complex
Licensing factor
Minichromosome maintenance
DNA re-replication
S phase
Replication factor C
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Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle
ABSTRACT DNA replication is a pivotal event in the cell cycle and, as a consequence, is tightly controlled in eukaryotic cells. The initiation of DNA replication is dependent upon the completion of mitosis and upon the commitment to complete the cell cycle made during G1. Characterisation of the protein factors required for initiating DNA replication is essential to understand how the cell cycle is regulated. Recent results indicate that initiation complexes assemble in multiple stages during the cell cycle. First, origins are bound by the multisubunit origin recognition complex (ORC) which is essential for DNA replication in vivo. ORC, present at little more than one complete complex per replication origin, binds to origins immediately after initiation in the previous cell cycle. ORC binding occurs by the recognition of a bipartite sequence that includes the essential ARS consensus sequence (ACS) and the functionally important Bl element adjacent to the ACS. A novel pre-replicative complex (pre-RC) assembles at origins at the end of mitosis in actively cycling cells and remains at origins until DNA replication initiates. Finally, Dbf4, which is periodically synthesised at the end of Gi, interacts with replication origins. Dbf4-origin interaction requires an intact ACS strongly suggesting that interaction occurs through ORC. Dbf4 interacts with and is required for the activation of the Cdc7 protein kinase and together, Dbf4 and Cdc7 are required for the Gi-S transition. Separate regions of Dbf4 are required for Cdc7- and origin-interaction suggesting that Dbf4 may act to recruit Cdc7 to replication origins where phosphorylation of some key component may cause origin firing.
Origin recognition complex
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Pre-replication complex
Minichromosome maintenance
DNA re-replication
S phase
Replication factor C
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Abstract The budding yeast Cdc6 protein is important for regulating DNA replication intiation. Cdc6p acts at replication origins, and cdc6‐1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc 18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper (1) further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G 1 , and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.
Origin recognition complex
Minichromosome maintenance
Pre-replication complex
DNA re-replication
Replication factor C
Licensing factor
S phase
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In all eukaryotes, it is essential that cells maintain a strict control over S phase to ensure that DNA replication occurs once, and only once, per cell division cycle. To achieve this, cells must promote DNA replication during the G1 phase and then repress DNA replication during the G2 phase. In the fission yeast Schizosaccharomyces pombe, two proteins required for the initiation of DNA replication, Cdcl8 and Cdt1, may be important for this control over DNA replication. Cdcl8 and Cdt1 are both upregulated in G1 and have an essential function in initiation, and are then efficiently downregulated in G2. In this thesis, I have tested whether the downregulation of these initiation factors is important to repress DNA synthesis in G2. I have found that the accumulation of high levels of Cdcl8 are sufficient to induce origin firing in G2 cells and drive re-replication in the absence of mitosis. This re-replication is potentiated by the co-expression of Cdt1, which may be mediated by the stabilisation of Cdc18 on chromatin by Cdt1. Biochemical characterisation of these re-initiation events revealed that certain aspects of this re-replication are similar to those observed in wild type S phase cells, such as the requirement for the MCMs and the cyclin-dependent kinase Cdc2. However, these cells fail to undergo complete and efficient replication of the chromosomes suggesting that Cdc18 and Cdt1 can override the normal controls that block initiation, but this mechanism does not fully mimic initiation in a G1 cell. The findings presented in this thesis have identified and characterised multiple, overlapping mechanisms that downregulate Cdc18 and Cdt1 in G2. If these mechanisms are disrupted and Cdc18 and Cdt1 are allowed to accumulate in G2, replication competence can be re-established and cells re-replicate. Therefore, Cdc18 and Cdt1 form the core of the control which licenses chromosomes for replication.
Origin recognition complex
Licensing factor
Pre-replication complex
DNA re-replication
S phase
Replication factor C
Minichromosome maintenance
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Origin recognition complex
DNA re-replication
Licensing factor
Pre-replication complex
S phase
Replication factor C
Cite
Citations (97)
CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.
Origin recognition complex
Pre-replication complex
Licensing factor
DNA re-replication
Minichromosome maintenance
S phase
Replication factor C
Semiconservative replication
Replication timing
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The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4Cdt2) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.
Origin recognition complex
Minichromosome maintenance
Pre-replication complex
DNA re-replication
Licensing factor
S phase
Replication factor C
Cite
Citations (15)
Minichromosome maintenance
Origin recognition complex
S phase
Replication factor C
Pre-replication complex
Licensing factor
Semiconservative replication
Cite
Citations (22)