DNA Replication Licensing System Research Articles

Function of the amino-terminal region of human MCM4 in helicase activity.

The amino-terminal region of eukaryotic MCM4 is characteristic of the presence of a number of phosphorylation sites for CDK and DDK, suggesting that the region plays regulatory roles in the MCM2-7 helicase function. However, the roles are not fully understood. We analyzed the role of the amino-terminal region of human MCM4 by using MCM4/6/7 helicase as a model for MCM2-7 helicase. First we found that deletion of 35 amino acids at the amino-terminal end resulted in inhibition of DNA helicase activity of the MCM4/6/7 complex. Conversion of arginine at amino acid no. 10 and 11 to alanine had similar effect to the deletion mutant of Δ1-35, suggesting that these arginine play a role in the DNA helicase activity. The data suggest that expression of these mutant MCM4 in HeLa cells perturbed the progression of the S phase. Substitution of six CDK phosphorylation sites (3, 7, 19, 32, 54 and 110) in the amino-terminal region by phospho-mimetic glutamic acids affected the hexamer formation of the MCM4/6/7 complex. MCM4 phosphorylation by CDK may play a role in DNA replication licensing system, and the present results suggest that the phosphorylation interferes MCM function by lowering stability of MCM complex.

MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2–7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the “MCM paradox.” Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.

DNA replication licensing.

The DNA replication licensing system ensures that chromosomal DNA is replicated precisely once before cell division occurs. A DNA helicase must be loaded on origin DNA for replication to initiate. Considerable evidence suggests that the MCM complex acts as a replicative helicase in eukaryotes. When the MCM complex is loaded on the chromatin, the replication origin is formally defined as being licensed for replication. Licensing takes place several hours before origins are activated to undergo replication in S-phase. Genetic and biochemical studies show that the licensing process is well conserved in eukaryotes. Cyclin Dependent Kinases (CDKs), the master regulators of the cell cycle, coordinate the initiation of the two key cell cycle events, replication of DNA and its segregation at mitosis. Eukaryotes have developed complex regulatory mechanisms to ensure that origin licensing is coordinated with these events so that genome integrity is preserved during successive cell divisions.

Control of chromosomal DNA replication in the early Xenopus embryo.

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Evidence for Different MCM Subcomplexes with Differential Binding to Chromatin inXenopus

MCM proteins are molecular components of the DNA replication licensing system inXenopus.These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes. We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-boundXenopusMCM proteins during the cell cycle. In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones. The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control. Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits. Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3. Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes.

Sequence of mouse CDC47, a member of the minichromosome maintenance (Mem) family involved in the DNA replication licensing system

The cDNA encoding a mouse protein, mCDC47, belonging to the minichromosome maintenance (Mcm) family possibly involved in the DNA replication licensing system, was cloned and seqeuced. Its deduced amino acid (aa) sequence contained a putative DNA-dependent ATPase motif commonly observed in the family, and a zinc-finger-like domain found in some members of the family.

Purification of an MCM-containing complex as a component of the DNA replication licensing system

Replication licensing factor (RLF) ensures that eukaryotic chromosomal DNA is replicated exactly once in each cell cycle. On exit from metaphase, RLF is activated and binds to or modifies chromatin. This modification (the 'licence') is required for subsequent DNA replication; the licence is also inactivated in the process of replication. Active RLF is not imported into the nucleus, so further DNA replication cannot occur until the DNA is relicensed by passage throught mitosis. We have developed an assay to purify RLF from Xenopus eggs. Activity resolves into two components, RLF-M and RLF-B, both of which are required for licensing. RLF-M has been purified to apparent homogeneity: it consists of three polypeptides, one of which is a Xenopus homologue of the yeast MCM3 protein. Xenopus Mcm3 associates with chomatin in G1 and is removed during replication, consistent with its being a component of the RLF system.