Clb 5,6 (Cdk1)

Last updated March 20, 2019

Clb5 and Clb6 are B-type, S-phase cyclins in yeast that assist in cell cycle regulation. [1] Clb5 and Clb6 bind and activate Cdk1, and high levels of these cyclins are required for entering S-phase. [2] S-phase cyclin binding to Cdk1 directly stimulates DNA replication as well as progression to the next phase of the cell cycle. [3]

Yeasts are eukaryotic single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and 1,500 species are currently identified. They are estimated to constitute 1% of all described fungal species. Yeasts are unicellular organisms which evolved from multicellular ancestors, with some species having the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae. Yeast sizes vary greatly, depending on species and environment, typically measuring 3–4 µm in diameter, although some yeasts can grow to 40 µm in size. Most yeasts reproduce asexually by mitosis, and many do so by the asymmetric division process known as budding.

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the basis for biological inheritance. The cell possesses the distinctive property of division, which makes replication of DNA essential.

Structure

Clb5 and Clb6 are two of the six B-type cyclins in budding yeast, which contain a short, hydrophobic amino acid sequence that allows targeted degradation and phosphorylation of some proteins that regulate DNA replication. This degradation occurs in late mitosis and is regulated by the anaphase promoting complex (APC). [1] Clb1-6 all target and activate the single yeast cyclin-dependent kinase, Cdk1. [1]

Saccharomyces cerevisiae is a species of yeast. It has been instrumental to winemaking, baking, and brewing since ancient times. It is believed to have been originally isolated from the skin of grapes. It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like Escherichia coli as the model bacterium. It is the microorganism behind the most common type of fermentation. S. cerevisiae cells are round to ovoid, 5–10 μm in diameter. It reproduces by a division process known as budding.

Clb6 is encoded by 380 amino acids (44.1kDa), and is 49.7% identical to Clb5. [2] Clb5 is 435 amino acids (50.4 kDa). [2] The hydrophobic box motif is found on the C terminus of both cyclins, and includes the conserved FLRRISK sequence characterizing B-type cyclins. [2]

Function

Clb5 and Clb6 are part of a regulatory network that initiates DNA replication during S-phase. Clb5 and Clb6 levels rise during G1 (earlier than other B-type cyclins) and stay high throughout S and M phases. [1]

The g₁ phase, or Gap 1 phase, is the first of four phases of the cell cycle that takes place in eukaryotic cell division. In this part of interphase, the cell synthesizes mRNA and proteins in preparation for subsequent steps leading to mitosis. G₁ phase ends when the cell moves into the S phase of interphase.

During S-phase, Clb5 and Clb6 are simultaneously expressed with other genes encoding proteins required for individual DNA strand replication and separation. [1] Clb5 and Clb6 differentially bind to Cdk1, and this activation directly promotes firing of the various origins of replication. [1] Clb5, in particular, has unique hydrophobic section of amino acids that allows specific interactions with proteins in the pre-replication complex bound on the DNA and helps localize Clb5 to DNA replication origins.

A pre-replication complex (pre-RC) is a protein complex that forms at the origin of replication during the initiation step of DNA replication. Formation of the pre-RC is required for DNA replication to occur. Complete and faithful replication of the genome ensures that each daughter cell will carry the same genetic information as the parent cell. Accordingly, formation of the pre-RC is a very important part of the cell cycle.

Clb5 and Clb6 also assist in spindle pole body duplication during S-phase, primarily when Clb3 and Clb4 are inactivated. [2] However, the spindle pole body duplication and its interactions with Clb5 are not well understood. In contrast to the other B-type cyclins, that negatively regulate SCB-binding factor (SBF) and MCB-binding factor (MBF), [1] [2] Clb5 and Clb6 can activate the G1/S transition in the absence of the G1 cyclins Cln1,2,3. These gene regulatory proteins control G1/S genes, and their negative regulation assists in shutting off expression of G1 cyclins during S-phase. Finally, Clb5-Cdk1 has been shown to be important for the phosphorylation of Cdh1 in yeast, and Clb5 destruction promotes dephosphorylation of Sic1. [1] Destruction of Clb5 and Clb6 is usually mediated by APC-Cdc20. [1] Studies have also shown that cells lacking Clb5 and Clb6 have dramatically reduced sporulation efficiencies.

The spindle pole body (SPB) is the microtubule organizing center in yeast cells, functionally equivalent to the centrosome. Unlike the centrosome the SPB does not contain centrioles. The SPB organises the microtubule cytoskeleton which plays many roles in the cell. It is important for organising the spindle and thus in cell division.

Cdh1 is one of the substrate adaptor protein of the anaphase-promoting complex (APC) in the budding yeast Saccharomyces cerevisiae. Functioning as an activator of the APC/C, Cdh1 regulates the activity and substrate specificity of this ubiquitin E3-ligase.

Sic1, a protein, is a stoichiometric inhibitor of Cdk1-Clb complexes in the budding yeast Saccharomyces cerevisiae. Because B-type cyclin-Cdk1 complexes are the drivers of S-phase initiation, Sic1 prevents premature S-phase entry. Multisite phosphorylation of Sic1 is thought to time Sic1 ubiquitination and destruction, and by extension, the timing of S-phase entry.

Interactions

Sic1 Regulation

Clb5 and Clb6 levels are high at the beginning of S-phase, though they initially rise in G1. Upon commitment to cell division, G1/S cyclin levels rise, bind Cdk1, and immediately form active complexes. Clb5 and Clb6 are also expressed and bind Cdk1, but are inactive based on control from the Clb-specific Cdk1 inhibitor Sic1. [1] G1/S cyclin-Cdk complexes promote the destruction of Sic1 and allow activation of Clb5- and Clb6-Cdk1 complexes. [1]

As the yeast cell transitions through G1, there is a large, inactive pool of Clb5 and Clb6-Cdk1 complexes. After activation, Clb5 and Clb6 can stimulate DNA replication, but also phosphorylate Sic1, targeting it for destruction. [1] Thus, Clb5 and Clb6 are engaged in a positive feedback loop to promote their own activation during this period of the cell cycle. [1]

APC Interaction

An important regulatory event during G1 is the inactivation of the anaphase promoting complex (APC-Cdh1). [1] Clb5 and Clb6 activation assists in APC-Cdh1 inactivation, although the complete mechanism is unclear. It is hypothesized that Clb5 and Clb6 are somewhat resistant to APC-Cdh1 degradation since they are primarily regulated by APC-Cdc20. [1]

Mutations

Mitosis

There are specific origins of replication that are activated in either the early or late stage of the cell cycle. Directed mutational studies targeting the Clb5 and Clb6 encoding genes have shown that both can activate origins usually replicated early in the cell cycle, but only Clb5 can activate late-stage origins. In cells without Clb5, S-phase is extended because late-stage origins are required to be replicated through the gradual spread of replication forks rather than Clb5-stimulated replication. [1] [4] Cells without Clb6 have little to no phenotypic changes since Clb5 can activate both types of origins. [4] In double mutants for Clb5 and Clb6, the onset of S-phase (rather than the length) is significantly delayed, but will eventually occur as a side result of the buildup of other mitotic cyclins (Clb1-4). [4] S-phase length is normal in these double mutants. [2]

Meiosis

Similar studies have shown a significant difference between mitosis and meiosis progression in cells lacking Clb5 or Clb6, primarily that meiosis S-phase cannot occur properly without Clb5. These mutant cells segregate their unreplicated DNA, which is lethal, and fail to activate the MEC1-DNA M-phase checkpoint, which usually inhibits cell cycle progression if DNA has not been replicated. [1] [3] The inability of Clb1-4 to compensate for the lack of Clb5 activity could potentially be explained by timing and accumulation arguments. In this hypothesis, cyclin concentrations must rise and accumulate to proceed to the next stage of the cell cycle. In mitotic growth, Clb1-4 levels rise immediately after Clb5 and Clb6 levels, allowing rapid accumulation. In meiotic growth, the interval before Clb1-4 levels rise following Clb5 and Clb6 expression is lengthened, allowing less time for accumulation and the resulting high levels required for recovery. [3] There is also evidence that Clb5 mutant cells are less likely to have DNA recombinations from double strand breaks (DSB), which may be a side effect of this Clb5 regulation. [5]

Differences between Clb5 and Clb6

There are “subtle and poorly understood” functional differences between these two cyclins. [1] Clb5 activates early and late phase origins of replication, whereas Clb6 only activates late phase origins. Functionally, Clb5 also has a mitotic destruction box that has been implicated in various proteolytic functions. Clb5 has also been shown to rescue Cln1-3 triple mutants, which is unique among B-type yeast cyclins. [4]

Related Research Articles

The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to duplication of its DNA and division of cytoplasm and organelles to produce two daughter cells. In bacteria, which lack a cell nucleus, the cell cycle is divided into the B, C, and D periods. The B period extends from the end of cell division to the beginning of DNA replication. DNA replication occurs during the C period. The D period refers to the stage between the end of DNA replication and the splitting of the bacterial cell into two daughter cells. In cells with a nucleus, as in eukaryotes, the cell cycle is also divided into two main stages: interphase and the mitotic (M) phase. During interphase, the cell grows, accumulating nutrients needed for mitosis, and undergoes DNA replication preparing it for cell division. During the mitotic phase, the replicated chromosomes and cytoplasm separate into two new daughter cells. To ensure the proper division of the cell, there are control mechanisms known as cell cycle checkpoints.

Anaphase-promoting complex is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin (Apc2) and RING (Apc11) subunit much like SCF. Other parts of the APC/C still have unknown functions, but are highly conserved.

A cyclin-dependent kinase complex is a protein complex formed by the association of an inactive catalytic subunit of a protein kinase, cyclin-dependent kinase (CDK), with a regulatory subunit, cyclin. Once cyclin-dependent kinases bind to cyclin, the formed complex is in an activated state. Substrate specificity of the activated complex is mainly established by the associated cyclin within the complex. Activity of CDKCs is controlled by phosphorylation of target proteins, as well as binding of inhibitory proteins.

S phase (Synthesis Phase) is the phase of the cell cycle in which DNA is replicated, occurring between G₁ phase and G₂ phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.

G₂ phase, or Gap 2 phase, is the second subphase of Interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G₂ phase ends with the onset of prophase, the first phase of mitosis in which the cell’s chromatin condenses into chromosomes.

Endoreduplication is replication of the nuclear genome in the absence of mitosis, which leads to elevated nuclear gene content and polyploidy. Endoreplication can be understood simply as a variant form of the mitotic cell cycle (G1-S-G2-M) in which mitosis is circumvented entirely, due to modulation of cyclin-dependent kinase (CDK) activity. Examples of endoreplication characterized in arthropod, mammalian, and plant species suggest that it is a universal developmental mechanism responsible for the differentiation and morphogenesis of cell types that fulfill an array of biological functions. While endoreplication is often limited to specific cell types in animals, it is considerably more widespread in plants, such that polyploidy can be detected in the majority of plant tissues.

Cell cycle checkpoints are control mechanisms in eukaryotic cells which ensure proper division of the cell. Each checkpoint serves as a potential point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring when favorable conditions are met. Currently, there are three known checkpoints: the G1 checkpoint, also known as the restriction or start checkpoint or ; the G2/M checkpoint; and the metaphase checkpoint, also known as the spindle checkpoint.

The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is a cell cycle check point where DNA integrity is assessed and the cell cycle can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated check point can lead to cellular transformation and disease states such as cancer

Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages.

Cyclin-dependent kinase 1 also known as CDK1 or cell division cycle protein 2 homolog is a highly conserved protein that functions as a serine/threonine kinase, and is a key player in cell cycle regulation. It has been highly studied in the budding yeast S. cerevisiae, and the fission yeast S. pombe, where it is encoded by genes cdc28 and cdc2, respectively. In humans, Cdk1 is encoded by the CDC2 gene. With its cyclin partners, Cdk1 forms complexes that phosphorylate a variety of target substrates ; phosphorylation of these proteins leads to cell cycle progression.

Cdc6, or cell division cycle 6, is a protein in eukaryotic cells that is studied in the budding yeast Saccharomyces cerevisiae. It is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle that coordinate S phase and mitosis. It is part of the pre-replicative complex (pre-RC) and is required for loading minichromosome maintenance (MCM) proteins onto the DNA, an essential step in the initiation of DNA synthesis. In addition, it is a member of the family of AAA+ ATPases and highly associated to Orc1p.

A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.

Cdc14 and Cdc14 are a gene and its protein product respectively. Cdc14 is found in most of the eukaryotes. Cdc14 was defined by Hartwell in his famous screen for loci that control the cell cycle of Saccharomyces cerevisiae. Cdc14 was later shown to encode a protein phosphatase. Cdc14 is dual-specificity, which means it has serine/threonine and tyrosine-directed activity. A preference for serines next to proline is reported. Many early studies, especially in the budding yeast Saccharomyces cerevisiae, demonstrated that the protein plays a key role in regulating late mitotic processes. However, more recent work in a range of systems suggests that its cellular function is more complex.

In cell biology, eukaryotes possess a regulatory system that ensures that DNA replication occurs only once per cell cycle.

Cln1, Cln2, and Cln3 are cyclin proteins expressed in the G1-phase of the cell cycle of budding yeast. Like other cyclins, they function by binding and activating cyclin-dependent kinase. They are responsible for initiating entry into a new mitotic cell cycle at Start. As described below, Cln3 is the primary regulator of this process during normal yeast growth, with the other two G1 cyclins performing their function upon induction by Cln3. However, Cln1 and Cln2 are also directly regulated by pathways sensing extracellular conditions, including the mating pheremone pathway.

Mitotic Exit is an important transition point that signifies the end of mitosis and the onset of new G1 phase for a cell, and the cell needs to rely on specific control mechanisms to ensure that once it exits mitosis, it never returns to mitosis until it has gone through G1, S, and G2 phases and passed all the necessary checkpoints. Many factors including cyclins, cyclin-dependent kinases (CDKs), ubiquitin ligases, inhibitors of cyclin-dependent kinases, and reversible phosphorylations regulate mitotic exit to ensure that cell cycle events occur in correct order with fewest errors. The end of mitosis is characterized by spindle breakdown, shortened kinetochore microtubules, and pronounced outgrowth of astral (non-kinetochore) microtubules. For a normal eukaryotic cell, mitotic exit is irreversible.

DNA re-replication is an undesirable and possibly fatal occurrence in eukaryotic cells in which the genome is replicated more than once per cell cycle. Rereplication is believed to lead to genomic instability and has been implicated in the pathologies of a variety of human cancers. To prevent rereplication, eukaryotic cells have evolved multiple, overlapping mechanisms to inhibit chromosomal DNA from being partially or fully rereplicated in a given cell cycle. These control mechanisms rely on cyclin-dependent kinase (CDK) activity. DNA replication control mechanisms cooperate to prevent the relicensing of replication origins and to activate cell cycle and DNA damage checkpoints. DNA rereplication must be strictly regulated to ensure that genomic information is faithfully transmitted through successive generations.

The anaphase- promoting complex or cyclosome (APC/C) is a highly specific ubiquitin protein ligase responsible for triggering events of late mitosis. In early mitosis, Cdc20 levels rise and APC/C binds to form active APC/CCdc20. This then leads to the destruction of mitotic cyclins, securin, and other proteins to trigger chromosome separation in anaphase.In early anaphase, Cdk1 is inactivated, leading to the activation of Cdh1, the other activator subunit of APC/C. This then triggers the degradation of Cdc20 and leads to the activation of APC/CCdh1 through G1 to suppress S- phase cyclin-Cdk activity. At the end of G1, APC/CCdh1 is inactivated and S- phase and mitotic cyclins gets reaccumulate as the cell progresses to S phase.

References

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Morgan, DO (2007) 'The Cell Cycle: Principles of Control, Oxford University Press
1 2 3 4 5 6 7 Schwob, E. and K. Nasmyth (1993). "CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae." Genes & development 7(7a): 1160.
1 2 3 Stuart, D. and C. Wittenberg (1998). "CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint." Genes & development 12(17): 2698.
1 2 3 4 Epstein, C. B. and F. R. Cross (1992). "CLB5: a novel B cyclin from budding yeast with a role in S phase." Genes & development 6(9): 1695.
↑ Smith, K. N., A. Penkner, et al. (2001). "B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis." Current Biology 11(2): 88-97.

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[Morgan-1] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Morgan, DO (2007) 'The Cell Cycle: Principles of Control, Oxford University Press

[SE-2] 1 2 3 4 5 6 7 Schwob, E. and K. Nasmyth (1993). "CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae." Genes & development 7(7a): 1160.

[Stuart-3] 1 2 3 Stuart, D. and C. Wittenberg (1998). "CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint." Genes & development 12(17): 2698.

[Epstein-4] 1 2 3 4 Epstein, C. B. and F. R. Cross (1992). "CLB5: a novel B cyclin from budding yeast with a role in S phase." Genes & development 6(9): 1695.

[5] Smith, K. N., A. Penkner, et al. (2001). "B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis." Current Biology 11(2): 88-97.