Eukaryotic Replication Flashcards
similarities between Prokaryotic vs Eukaryotic Replication
Replication is mostly very similar
* Initiation must be regulated - the entire genome must be copied once during replication
* Bi-directional == starting from a specific point on the DNA (the origin of replication), the replication machinery works both “upstream” and “downstream” to replicate the DNA as efficiently as possible.
* Primase lays down RNA primers for DNA polymerase == primers provide the starting point for DNA synthesis.
* Highly processive polymerases do most of the DNA synthesis == thet can start adding nucleotides, they stay attached and add many nucleotides without falling off.
* DNA polymerases have 3’ to 5’ exonuclease activity (proofreading) == The 3’ to 5’ exonuclease activity refers to the ability of the DNA polymerase to move backwards on the strand and remove incorrect nucleotides, a process known as “proofreading”..
* Leading and lagging strands
Primase role
RNA primer are produced by the enzyme, Primase
RNA primers is short piece of RNA or DNA
provides the initial starting point with a 3’ OH group for the DNA polymerase to begin adding nucleotides to during DNA replication.
==> DNA polymerase needs the 3’OH group as the spot to add the next nucleotide (brick). Without the primer, the DNA polymerase wouldn’t know where to start building
the Eukaryotic Cell Cycle
- G1 (Gap 1): During this phase, the cell grows and prepares for DNA replication. The cell has the option to either enter the S phase (where DNA synthesis happens) or go into G0.
- G0: This is a phase where cells are not actively preparing to divide. Cells in this phase may be in a state of dormancy or they may be carrying out their regular functions without preparing for division.
Restriction Point: This is the “point of no return” in the cell cycle. Once a cell passes this point, it is committed to dividing. - S (Synthesis): During this phase, DNA replication occurs. Each chromosome is duplicated to ensure that the two new cells will each have a complete set of chromosomes.
- G2 (Gap 2): The cell prepares for mitosis by synthesizing proteins and growing in size.
- M (Mitosis): The cell divides its copied DNA and cytoplasm to create two new cells.
Counting Dividing Cells
One way to estimate the number of cells at each stage of the cell cycle is to analyze their DNA content. Cells in the G1 phase have a certain amount of DNA, cells in the S phase will have more as they are in the process of duplicating their DNA, and cells in G2 and M phases will have twice as much DNA as those in G1 because they have fully duplicated their DNA in preparation for cell division.
G0 phase
The G0 phase is a state in the cell cycle where cells are not preparing for division or actively dividing. During the G0 phase, cells carry out their normal functions and may undergo specialized processes like differentiation. Some cells, such as mature neurons or muscle cells, can remain in the G0 phase permanently. However, other cells can be induced to leave the G0 phase and re-enter the cell cycle in response to certain signals.
- % of cells in G0 increases with age: As an organism ages, a larger percentage of its cells enter the G0 phase and stop dividing. This can be due to various factors like accumulated DNA damage over time, reduced regenerative capacity, etc.
- Also differs between cell types: The propensity for cells to enter G0 varies greatly depending on the type of cell. Examples given are:
* Fibroblasts, epithelium (role in wound healing): These cell types are almost never in G0, meaning they are usually in a cycle of growth and division to help repair and maintain tissues.
* Adult liver cell: These cells enter the cell cycle approximately once a year. The rest of the time, they stay in the G0 phase.
* Adult brain cells: These cells are almost always in G0, which means they are not actively dividing. This is one reason why brain injuries or neurodegenerative diseases can be so detrimental; the brain has a limited ability to repair itself by creating new cells. - Quiescent cells: These are cells that are in a state of dormancy or G0 phase, but can be induced to re-enter the cell cycle by a mitotic signal. A mitotic signal is a kind of “wake-up call” that stimulates the cell to prepare for division.
- Senescent cells: These are aged cells that have lost the ability to divide and cannot re-enter the cell cycle. Senescence can occur after many rounds of cell division or due to DNA damage or metabolic stress.
CDKs in the Cell Cycle
cyclin-dependent protein kinases (CDKs) == protein kinases that play crucial roles in regulating cell cycle progression
- CDK activity is dependent on levels of cyclins (the two proteins associate): This means that the activity of CDKs, the enzymes that help control the cell cycle, is dependent on the presence of another type of protein called cyclins. The levels of cyclins in the cell fluctuate throughout the cell cycle, and their association with CDKs is what activates the CDKs.
- Changes in cyclin levels regulate progression through the cell cycle: As the levels of cyclins change, they control the activity of CDKs and thus help regulate the progress of the cell through the different phases of the cell cycle.
- Active CDKs phosphorylate proteins involved in regulation of cell cycle: Once the CDKs are active, they add phosphate groups to other proteins in the cell, a process known as phosphorylation. This phosphorylation of specific proteins is what drives the progression of the cell cycle.
how levels of cyclin change :
New cyclins are synthesized (produced) by the cell during specific stages of the cell cycle. This synthesis is controlled at the level of gene expression, meaning that the cell controls when the genes for cyclins are “turned on” and new cyclins are produced.
Checkpoints in the Cell Cycle
- G1 to S checkpoint (where cells in This is the primary “decision point” where the cell senses its surroundings and decides whether to initiate DNA replication. The cell checks for DNA damage and adequate resources. If conditions are not favorable, the cell can enter a resting state (G0 phase).
- S to G2 checkpoint (all divided and can grow and produce proteins): This checkpoint ensures that DNA replication has completed properly and that no damage has occurred to the DNA during replication. If errors are detected, the cell cycle is halted, and the cell attempts to correct the errors.
- G2 to M checkpoint: This checkpoint confirms that DNA replication has occurred without any errors. It also ensures that the cell has properly prepared for mitosis by producing the necessary proteins.
- M checkpoint: This checkpoint ensures that sister chromatids (the two identical copies of a single chromosome that are joined together by a centromere) are correctly attached to the spindle fibers before the cell divides. Improper attachment could lead to an unequal distribution of chromosomes in the daughter cells (a situation called aneuploidy).
Checkpoint: G1 to S
Controlling progression through the cell cycle: The passage emphasizes the importance of tightly controlling the cell cycle progression. Before moving from G1 to S phase (DNA synthesis), the cell checks to make sure there’s no DNA damage. If damage exists, the cell will attempt to repair it before proceeding. Once DNA synthesis is complete, the cell ensures this before progressing to the G2 phase. Additionally, before the cell divides, it checks to make sure spindle fibers, which help segregate chromosomes during cell division, are correctly attached.
Phosphorylation of target proteins by active CDKs: Cyclin-dependent kinases (CDKs) are proteins that control the progression of the cell cycle. When they’re active, they can add a phosphate group (a process called phosphorylation) to other proteins, which can affect these proteins’ functions. For example, phosphorylation can activate other proteins (such as other kinases) or affect the way proteins interact with each other. This process is a key part of how the cell controls its progression through the cell cycle.
==> the cell carefully controls its progression through the cell cycle to ensure that everything occurs in the right order and under the right conditions.
Control of Cell Division in Eukaryotes
- Proto-oncogenes control cell growth
–> Oncogenes – the accelerator: Oncogenes are mutated versions of normal genes, called proto-oncogenes, that control cell growth. When a proto-oncogene is functioning normally, it helps regulate cell growth and prevents cells from growing and dividing too rapidly or in an uncontrolled way. However, when a proto-oncogene mutates (changes) or there are too many copies of it, it becomes an oncogene and can promote cell growth and division at an accelerated, uncontrolled rate.
Mutations are dominant – one copy is enough to be stuck on the accelerator = These mutations are dominant, meaning that only one copy of the gene needs to be altered to influence cell growth
- Tumour suppressor genes – the brakes: These are like the body’s natural brake system on cell division. When functioning normally, tumor suppressor genes slow down cell division, repair DNA mistakes, or tell cells when to die (a process known as apoptosis or programmed cell death). When these genes mutate, they can lose their braking ability, which can lead to uncontrolled cell growth—a key characteristic of cancer. Unlike oncogenes, tumor suppressor gene mutations are often recessive, meaning both copies of the gene (one from each parent) need to be altered for an effect to be seen.
- Unregulated cell division → cancer: When the mechanisms controlling cell division and growth are disrupted (as in the case of malfunctioning oncogenes or tumor suppressor genes), cells can begin to divide uncontrollably. This unregulated cell growth can lead to the development of a tumor or cancer.
DNA changes and cancer
How certain DNA changes or mutations can lead to cancer. These mutations can either be inherited or acquired:
- Inherited mutations: Some people inherit faulty versions of genes from their parents. The examples given, Rb and BRCA1, are both genes associated with a higher risk of certain cancers when mutated. Rb stands for Retinoblastoma, a gene that produces a tumor suppressor protein. Mutations in this gene can lead to the development of a rare form of cancer called retinoblastoma in children. BRCA1 is another gene, and mutations in it significantly increase the risk of breast and ovarian cancer.
- Acquired mutations: These are not inherited but occur at some point during a person’s life. They can occur randomly during normal cell division, or be provoked by external factors such as certain behaviours (smoking, for example) or exposure to radiation or certain chemicals.
Example : retinoblastoma protein
== this protein is a tumour suppressor, meaning under normal circumstances it helps regulate cell division and prevent the formation of tumours. However, when a child inherits one mutated copy of the Rb gene, and then acquires a mutation in the other copy in retinal cells, it can lead to retinoblastoma. Additionally, people with Rb mutations also have an increased risk of other types of cancer, including lung, prostate, and breast cancer.
Differences in Prokaryotic vs Eukaryotic replication
Eukaryotic replication is slower than prokaryotic replication: Replication in eukaryotes occurs at approximately 50 nucleotides per second, which is around 1/20th the rate of replication in the bacterium E. coli
In eukaryotic cells, DNA polymerase α forms a complex with primase and initiates DNA synthesis by laying down RNA primers. In contrast, prokaryotes uses to the DNA Polymerase I in and also has low processivity meaning it disassociates from the DNA quickly, i.e., it synthesizes short stretches of DNA before falling off, proofreading abilities
DNA polymerase δ is responsible for the bulk of DNA synthesis in eukaryotic cells. It’s highly processive, meaning it can synthesize long stretches of DNA before falling off. It performs a role similar to that of the DNA Polymerase III in prokaryotes
Amount of Genetic Material: Eukaryotes have much more genetic material to copy and package, necessitating many origins of replication (e.g., 30,000 to 50,000 in humans). Not all of these origins are activated in each round of replication, but enough are to ensure the entire chromosome is copied
DNA polymerase δ
For the bulk of DNA synthesis, eukaryotes use DNA polymerase δ, which is highly processive. This enzyme is responsible for the bulk of DNA synthesis in eukaryotic cells. It’s highly processive, meaning it can synthesize long stretches of DNA before falling off. It performs a role similar to that of the DNA Polymerase III in prokaryotes
Indeed, DNA Polymerase δ in eukaryotes does have proofreading abilities
DNA polymerase α
In eukaryotic cells, DNA replication begins with the formation of this complex. DNA polymerase α and primase work together to begin the process of DNA synthesis. Primase, an RNA polymerase, lays down a short RNA sequence called a primer. This primer provides the necessary 3’ OH group to which DNA polymerase α can add DNA nucleotides. This step is crucial because DNA polymerase can only add nucleotides to an existing chain of nucleotides
features:
* low processivity = means that after adding a few nucleotides to the growing DNA chain, it easily disassociates, or falls off, from the DNA template. This characteristic implies that DNA polymerase α synthesizes short stretches of DNA before it disconnects from the template
* No proofreading: Unlike many other DNA polymerases, DNA polymerase α lacks proofreading activity. In the context of DNA replication, proofreading refers to the ability of a DNA polymerase to identify and correct errors it makes while adding nucleotides to the growing DNA chain. Without this ability, any mistakes made by DNA polymerase α go uncorrected, which could potentially lead to mutations in the synthesized DNA
Eukaryotic Replication:
Yes, that’s right. The larger amount of genetic material in eukaryotes necessitates numerous origins of replication to ensure that the entire genome is copied within a reasonable timeframe. Not all origins are activated in each cycle, but enough are to ensure the entire chromosome is copied. The number of origins varies, with some estimates suggesting there are between 30,000 to 50,000 in humans. It’s also a regulated process so that replication occurs only once per cell cycle, which aids in the prevention of errors or aberrations
Initiation of cell cycle
G1 Phase: The cell selects origin sites and assembles pre-replicative complexes (pre-RCs) at these locations. These complexes are crucial for the initiation of DNA replication.
S Phase: Active cyclin-dependent kinases (CDKs) phosphorylate and activate pre-RCs, which then recruit DNA polymerases to the origin sites. Clusters of origin sites, typically numbering between 20 to 80, are initiated at a time. Additionally, CDKs inhibit the formation of new pre-RCs if replication has already started at an origin site. This regulation ensures DNA replication occurs only once per cell cycle at each origin
