Cell Cycle Checkpoints PDF

Cell cycle checkpoint Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed with progression through the variou...

Cell cycle checkpoint Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination 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 only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: The G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to- anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein. Phases of Cell Cycle A typical eukaryotic cell cycle is illustrated by human cells in culture. These cells divide once in approximately every 24 hours. However, this duration of cell cycle can vary from organism to organism and also from cell type to cell type. Yeast for example, can progress through the cell cycle in only about 90 minutes. The cell cycle is divided into two basic phases: - Interphase (cell growth and copying of chromosomes in preparation for cell division) - Mitotic (M) phase (mitosis and cytokinesis) A) Interphase 1 It represents continuous growth of the cell and is subdivided into three phases, G1 (gap1) phase, S (synthesis) phase and G2 (gap 2) phase. 1- The G1 phase It is usually the longest and the most variable phase of the cell cycle, and it begins at the end of M phase. During the G1 phase, the cell gathers nutrients and synthesizes RNA and proteins necessary for DNA synthesis and chromosome replication. 2- The S phase (DNA replication) Initiation of DNA synthesis marks the beginning of the S phase, which is about 7.5 to 10 hours in duration. The DNA of the cell is doubled during the S phase, and new chromatids are formed. 3- The G2 phase (cell preparation for cell division) During this phase, the cell examines its replicated DNA in preparation for cell division. This is a period of cell growth and reorganization of cytoplasmic organelles before entering the mitotic cycle. The G2 phase may be as short as 1 hour in rapidly dividing cells or of nearly indefinite duration in some polypoid cells and in cells such as the primary oocyte that are arrested in G2 for extended periods. B) M Phase (Mitosis phase) Mitosis nearly always includes both karyokinesis (division of the nucleus) and cytokinesis (division of the cell) and lasts about 1 hour. Mitosis takes place in several stages described in more detail below. Separation of two identical daughter cells concludes the M phase. Mitosis Cell division is a crucial process that increases the number of cells, permits renewal of cell populations, and allows wound repair. 2 Mitosis is a process of chromosome segregation and nuclear division followed by cell division that produces two daughter cells with the same chromosome number and DNA content as the parent cell. The process of cell division includes division of both the nucleus (karyokinesis) and the cytoplasm (cytokinesis). The process of cytokinesis results in distribution of nonnuclear organelles into two daughter cells. Before entering mitosis, cells duplicate their DNA in the S or synthesis phase. Phases of Mitosis 1. Prophase: The replicated chromatin condenses and become visible as chromosomes. Each chromosome can be seen to consist of two chromatids. The sister chromatids are held together by the ring of proteins at the centromere. In late prophase, the nuclear envelope begins to disintegrate, and the nucleolus completely disappears. In addition, a highly specialized protein complex called a kinetochore appears on each chromatid opposite to the centromere. 2. Metaphase: Formation of the mitotic spindle, consisting of three types of microtubules, that becomes organized around the centrosomes, the astral microtubules, the polar microtubules and the kinetochore microtubules. When a kinetochore is finally captured by a kinetochore microtubule, it is pulled toward the centrosomes, Kinetochore microtubules and their associated motor proteins direct the movement of the chromosomes to a plane in the middle of the cell, called the equatorial or metaphase plate. 3. Anaphase: Separation of sister chromatids. This separation occurs when the proteins that have been holding the chromatids together break down. The separated chromatids 3 are pulled to opposite poles of the cell by the sliding along the kinetochore microtubules toward the centrosomes. 4. Telophase: Reconstitution of a nuclear envelope around the chromosomes at each pole. The chromosomes uncoil and become indistinct. The nucleoli reappear, and the cytoplasm divides (cytokinesis) to form two daughter cells. Cytokinesis Cytokinesis begins with the furrowing of the plasma membrane midway between the poles of the mitotic spindle. The separation at the cleavage furrow is achieved by a contractile ring consisting of a very thin array of actin filaments positioned around the perimeter of the cell. As the ring tightens, the cell is pinched into two daughter cells. Because the chromosomes in the daughter cells contain identical copies of the duplicated DNA, the daughter cells are genetically identical and contain the same kind and number of chromosomes. The daughter cells are (2d) in DNA content and (2n) in chromosome number. Meiosis Meiosis involves two sequential nuclear divisions followed by cell divisions that produce gametes (sex cells) containing half the number of chromosomes and half the DNA found in somatic cells. -The zygote (the cell resulting from the fusion of an ovum and a sperm) and all the somatic cells derived from it are diploid (2n) 4 in chromosome number (46 chromosomes in human); thus, their cells have two copies of every chromosome and every gene encoded on this chromosome. -These chromosomes are called homologous chromosomes because they are similar but not identical; one set of chromosomes is of maternal origin, the other is from paternal origin. - The gametes, having only one member of each chromosome pair, are described as haploid (1n). - During gametogenesis, reduction in chromosome number to the haploid state (23 chromosomes in humans) occurs through meiosis. - This reduction is necessary to maintain a constant number of chromosomes in a given species. - Reduction in chromosome number to (1n) in the first meiotic division is followed by reduction in DNA content to the haploid (1d) amount in the second meiotic division. - During meiosis, the chromosome pair may exchange chromosome segments, thus altering the genetic composition of the chromosomes. This genetic exchange, called crossing-over, and the random assortment of each member of the chromosome pairs into haploid gametes give rise to infinite genetic diversity. Differences in Meiosis between Male & Female The nuclear events of meiosis are the same in males and females, but the cytoplasmic events are markedly different. In males, the two meiotic divisions of a primary spermatocyte yield four structurally identical, although genetically unique, haploid spermatids. Each spermatid has the capacity to differentiate into a spermatozoon. In contrast, in females, the two meiotic divisions of a primary oocyte yield one haploid ovum and three haploid polar bodies. The ovum receives most of the 5 cytoplasm and becomes the functional gamete. The polar bodies receive very little cytoplasm and degenerate. Divisions & Phases of Meiosis Meiosis consists of two successive mitotic divisions without the additional S phase between the two divisions. During the S phase that precedes meiosis, DNA is replicated forming sister chromatids (two parallel strands of DNA) joined together by the centromere. The DNA content becomes (4d), but the chromosome number remains the same (2n). The cells then undergo a reductional division (meiosis I) and an equatorial division (meiosis II). During meiosis I, as the name reductional division implies, the chromosome number is reduced from diploid (2n) to haploid (1n), and the amount of DNA is reduced from the (4d) to (2d). No DNA replication precedes meiosis II. The division during meiosis II is always equatorial because the number of chromosomes does not change. It remains at (1n), although the amount of DNA represented by the number of chromatids is reduced to (1d). Phases of Meiosis I 1. Prophase I: It is an extended phase that is subdivided into the following five stages: Leptotene: chromosomes start to condense. Zygotene: homologous chromosomes become closely associated (synapsis) to form pairs of chromosomes (bivalents) consisting of four chromatids (tetrads). Pachytene: crossing over between pairs of homologous chromosomes to form chiasmata (sing. chiasma). Diplotene: homologous chromosomes start to separate but remain attached by chiasmata. Diakinesis: homologous chromosomes continue to separate, and chiasmata move to the ends of the chromosomes. 6 2. Metaphase I: Metaphase I is similar to the metaphase of mitosis except that the paired chromosomes are aligned at the equatorial plate with one member on either side. - The chiasmata are cut, and the homologous chromosomes separate completely. - The spindle microtubules begin to interact with the chromosomes through the kinetochore at the centromere. - The chromosomes undergo movement to ultimately align their centromeres along the equatorial plate with one member of the homologous chromosomes on either side. 3. Anaphase I: The sister chromatids, held together by protein complexes and by the centromere, remain together. - A maternal or paternal member of each homologous pair moves to each pole. - Segregation or random assortment occurs because the maternal and paternal chromosomes of each pair are randomly aligned on one side or the other of the metaphase plate, thus contributing to genetic diversity. 4.Telophase I: - Homologous chromosomes, each consisting of two sister chromatids, are at the opposite poles of the cell. - Reappearance of the nucleolus and nuclear envelope. - At the completion of meiosis I, the cytoplasm divides. Each resulting daughter cell is haploid in chromosome number (1n) and contains one member of each homologous chromosome pair. The cell is still diploid in DNA content (2d). 7 Phases of Meiosis II: After meiosis I, the cells quickly enter meiosis II without passing through an S phase. - Meiosis II is an equatorial division and resembles mitosis. - During this phase, the sister chromatids will separate at anaphase II and move to opposite poles of the cell. - During meiosis II, the cells pass through prophase II, metaphase II, anaphase II, and telophase II. - These stages are essentially the same as those in mitosis except that they involve a haploid set of chromosomes (1n) and produce daughter cells that have only haploid DNA content (1d). - Unlike the cells produced by mitosis, which are genetically identical to the parent cell, the cells produced by meiosis are genetically unique. By the end of meiosis II, each parent cell (2n) give rise to 4 daughter cells with haploid number of chromosomes (In) and each daughter cell is genetically different from the parent cell 8 Chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. During mitosis and meiosis, chromatin facilitates proper segregation of the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this stage are the result of DNA being coiled into highly condensed chromatin. The total DNA in the cell is about 5 to 6 feet long which has to fit inside the nucleus of a cell in an orderly fashion. DNA molecules first wrap around the histone proteins forming beads on string structure called nucleosomes. Nucleosomes further coil and condense/gather to form fibrous material which is called chromatin. Chromatin fibers can unwind for DNA replication and transcription. When cells replicate, duplicated chromatins condense further to become a lot like chromosomes, visible under microscope which are separated into daughter cells during cell division. The overall structure of the chromatin network further depends on the stage of the cell cycle. During interphase, the chromatin is structurally loose to allow access to RNA and DNA polymerases that transcribe and replicate the DNA. The local structure of chromatin during interphase depends on the specific genes present in the DNA. Regions of DNA containing genes which are actively transcribed ("turned on") are less tightly compacted and closely associated with RNA polymerases in a structure known as euchromatin, while regions containing inactive genes ("turned off") are generally more condensed and associated with structural proteins in heterochromatin. Epigenetic modification of the structural proteins in chromatin via methylation and acetylation also alters local chromatin 9 structure and therefore gene expression. There is limited understanding of chromatin structure and it is active area of research in molecular biology. - Chromatin is the complex combination of DNA and proteins that makes up chromosomes. - It is found inside the nuclei of eukaryotic cells. - The function of chromatin is: -to package DNA into a smaller volume to get in the cell to strengthen the DNA to allow mitosis and meiosis. Types of Chromatin 1- Euchromatin 2- Heterochromatin 1-Euchromatin Euchromatin (also called "open chromatin") is a lightly packed form of chromatin (DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription. Euchromatin stands in contrast to heterochromatin, which is tightly packed and less accessible for transcription. 92% of the human genome is euchromatic. In eukaryotes, euchromatin comprises the most active portion of the genome within the cell nucleus. In prokaryotes, euchromatin is the only form of chromatin present; this indicates that the heterochromatin structure evolved later along with the nucleus, possibly as a mechanism to handle increasing genome size. Functions of euchromatin Euchromatin is the part of the chromatin involved in the active transcription of DNA into mRNA. As euchromatin is more open in order to allow the recruitment of RNA polymerase complexes and gene regulatory proteins, so transcription can be initiated. 10 There is a direct link between how actively productive a cell is and the amount of euchromatin in its nucleus. 2-Heterochromatin Heterochromatin is a tightly packed form of DNA or condensed DNA, which comes in multiple varieties. These varieties lie on a continuum between the two extremes of constitutive heterochromatin and facultative heterochromatin. Both play a role in the expression of genes. Because it is tightly packed, it was thought to be inaccessible to polymerases and therefore not transcribed.much of this DNA is in fact transcribed, but it is continuously turned over via RNA-induced transcriptional silencing (RITS). Heterochromatins are two types: - A- Constitutive heterochromatin - Constitutive heterochromatin domains are regions of DNA. - Found throughout the chromosomes of eukaryotes. - Heterochromatin is found at the pericentromeric regions of chromosomes but is also found at the telomeres and throughout the chromosomes. - Has a structural function.  Made up of satellite DNA. - Constitutive heterochromatin can affect the genes near itself (e.g. position- effect variegation). It is usually repetitive and forms structural functions such as centromeres or telomeres, in addition to acting as an attractor for other gene-expression or repression signals. B- Facultative heterochromatin 11 - In contrast facultative heterochromatin consists of euchromatin that takes on the staining and compactness characteristics of heterochromatin during same phase of development. - The inactive x-chromosomes is made up of facultative heterochromatin. - It may convert to euchromatin depending upon requirement. - Facultative heterochromatin is the result of genes that are silenced through a mechanism such as histone deacetylation or Piwi-interacting RNA (piRNA) through RNAi. It is not repetitive and shares the compact structure of constitutive heterochromatin. However, under specific developmental or environmental signalling cues, it can lose its condensed structure and become transcriptionally active. Functions of Heterochromatin Heterochromatin has been associated with several functions, from gene regulation to the protection of chromosome integrity; some of these roles can be attributed to the dense packing of DNA, which makes it less accessible to protein factors that usually bind DNA or its associated factors. For example, naked double-stranded DNA ends would usually be interpreted by the cell as damaged or viral DNA, triggering cell cycle arrest, DNA repair or destruction of the fragment, such as by endonucleases in bacteria. Some regions of chromatin are very densely packed with fibers that display a condition comparable to that of the chromosome at mitosis. Heterochromatin is 12 generally clonally inherited; when a cell divides, the two daughter cells typically contain heterochromatin within the same regions of DNA, resulting in epigenetic inheritance. Variations cause heterochromatin to encroach on adjacent genes or recede from genes at the extremes of domains. Transcribable material may be repressed by being positioned (in cis) at these boundary domains. This gives rise to expression levels that vary from cell to cell, which may be demonstrated by position-effect variegation. Insulator sequences may act as a barrier in rare cases where constitutive heterochromatin and highly active genes are juxtaposed (e.g. the 5'HS4 insulator upstream of the chicken β-globin locus, and loci in two Saccharomyces spp Nucleosome / Nucleosomes A nucleosome is a section of DNA that is wrapped around a core of proteins. Inside the nucleus, DNA forms a complex with proteins called chromatin, which allows the DNA to be condensed into a smaller volume. When the chromatin is extended and viewed under a microscope, the structure resembles beads on a string. Each of these tiny beads is a called a nucleosome and has a diameter of approximately 11 nm. The nucleosome is the fundamental subunit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. The chain of nucleosomes is then compacted further and forms a highly organized complex of DNA and protein called a chromosome. The DNA Packaging - DNA packaging. Each chromosome consists of one continuous thread-like molecule of DNA coiled tightly around proteins, and contains a portion of the 6,400,000,000 basepairs (DNA building blocks) that make up your DNA. The way DNA is packaged into chromatin is a factor in how protein production is controlled. 13 - Watson and Crick gave the DNA structure. According to their model DNA is a double-helical structure with two polynucleotide strands that run antiparallel. Due to phosphate groups in the DNA backbone, this double helix is negatively charged. The cell produces histone proteins that bind to the DNA to counteract the negative charge. These histone proteins have a role in the packaging of DNA. The Nucleosome and DNA Packaging - Although the DNA helical diameter is only 2 nm, the entire DNA strand in a single cell will stretch roughly 2 meters when completely unwound. The entire DNA strand must fit within the nucleus of a cell, so it must be very tightly packaged to fit. This is accomplished by wrapping the DNA around structural histone proteins, which act as scaffolding for the DNA to be coiled around. The entire structure is called a nucleosome, each of which includes an octamer of histone proteins and 146 to 147 base pairs of DNA. The millions of nucleosomes tightly coil the continuous DNA strand into chromatin which is further condensed into the chromosome classically visualized during cell division. - The tight structure of chromatin brings about the problem of accessibility to the DNA by enzymes involved in DNA replication and transcription. Chromatin exists in one of two states: heterochromatin, which is condensed and allows little access by transcription enzymes, and euchromatin, which is loose to allow for interaction with transcription enzymes. The transition between these two states is determined by interactions between the DNA and histone proteins via post-translational modifications to the histone proteins like methylation and acetylation. Methylation generally increases interactions between the DNA and histone, thus suppressing gene expression, whereas acetylation will loosen interactions resulting in greater access by transcription enzymes resulting in increased gene expression. The post-translational 14  modifications to histone proteins underlie the mechanisms of epigenetics, which are defined as alterations to gene expression without changes to the DNA sequence. - The ability for DNA packaging to be modified at various stages of the cell cycle is important in both DNA replication and cell division as well as transcription. Replication occurs at many origins of replication throughout the DNA strand to accelerate the replication of the entire genome, with each origin separated by approximately 100,000 base pairs. The DNA does not interact with histones during this process to allow for the propagation of the polymerase enzymes. However, when the process is complete, the DNA must reintegrate with the histones to reform nucleosomes and eventually the supercoiled chromosome structure during mitosis. Following cell division, the DNA must again separate from the histone proteins to undergo transcription. This capability for the DNA-histone interactions to be modulated is crucial for the proper growth and function of a cell with malfunctions contributing to disease like hypermethylation in cancer. 15