DNA synthesis & cell cycle Flashcards
The cell cycle consists of how many phases and what are they called?
2; Mitotic (M), and Interphase (cell growth and copying of chromosomes in preparation for cell division
Which phase consists of 90% of the cell cycle?
Interphase
G1 phase: first gap
S phase: synthesis
G2 phase: Second gap
G0 phase: Resting phase, postmitotic quiescent
G1 phase:
-1st gap phase
-Preparatory growth phase prior to cell entering DNA synthesis phase
-Cell is metabolically active; requires nutrients and growth factors, RNA/protein/lipid & carbohydrate synthesis occurs
-Many organelles are duplicated; No DNA replication yet
Duration: (6-24hrs); short in embryonic and cancer cells; Rapid or non-existent in rapidly dividing cells
S phase:
-DNA & chromosomal protein synthesis occurs
-Duration: approx 7-8 hours
-Cell is now committed to cell division; growth factors are no longer needed at this phase, DNA replication occurs here, creating two identical daughter genomes
G2 phase:
-2nd growth phase
-Interval between DNA synthesis (S phase) & mitosis (M phase)
-Enzyme, protein and ATP synthesis occurs (cell growth continues)
-Duration: lasts approx 3 hours
M phase:
-Mitotic phase
-Cell undergoes mitosis & then cytokinesis
Duration: 1-2 hours
What are the steps in the mitosis phase in order?
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
G0 phase:
-State of withdrawal from cell cycle
-Cell is neither dividing nor preparing to divide
-Instead, the cell is “doing its job”; performing it’s function within the tissue (common for differentiated cells)
-Examples of cells in G0; Hepatocytes, neurons
Checkpoints are based on series of _______________________ to initiate a specific cell-cycle events. What is this called?
biochemical switches
-Cell cycle control system
What are the three features of the biochemical switches?
1) Generally binary (on/off) to launch an event in a complete & irreversible fashion
2) Robust & Reliable; Contains back up mechanisms to ensure efficacy under variable conditions & if some components fail
3) Adaptable & modified to suit specific cell types (Responds to specific intracellular or extracellular signals); Cyclin dependant kinases (Cdks)
Points in the eukaryotic cell division cycle where progress through the cycle can be halted until conditions are suitable for the cell to proceed to the next stage:
Checkpoints “Transitions”
Checkpoints can be regulated by:
-Factors within the cell, mostly controlled by the “health” or “state of preparation” of the cell.
-Factors from outside the cell (messages from other cells within the same tissue or distant cells.
What are the three major regulatory transitions?
1) Start transition (aka G1/S)
2) G2/M transition
3) Metaphase to anaphase transition (aka M-to-A)
For most cells; G1/S seems to be the rate-limiting & committing step of the cell cycle
Cell signals are not necessary for a cell to pass through check points. (True/False)
False: all checkpoints rely on cell signals to keep the cell cycle moving. If these signals are not present, they will stop.
If there is a problem with completion of DNA replication, the cell will be held at what check point?
G2/M checkpoint
What is the key to cell cycle control sytem?
Cyclin-dependent kinases (Cdks); Controlled by a group of proteins called-Cyclins
-Correct and functional cyclin- cdk complexes are needed to progress through a checkpoint
Cdks are responsible for cyclical changes in _______________ of intracellular proteins that initiate/regulate the major events of the cell cycle.
phosphorylation
What causes the cyclic assembly and activation of cyclin-cdk complexes at specific stages of the cell?
Cyclical changes in cyclin protein levels
What are the 4 classes of cyclins that form specific complexes with cdks?
1) G1 cyclins: Cyclin D
2) G1/S cyclins: Cyclin E
3) S cyclins: Cyclin A
4) M cyclins: Cyclin B
What does G1 cyclins: Cyclin D do?
-Forms complex w/ cdk4 or cdk6
-Involved in G1 phase of cell cycle, needed for initiation of transcription of G1/S cyclins to help promote passage through the start transition
What do G1/S cyclins: Cyclin E do?
-Forms complex w/ Cdk2
-Bind Cdk’s at the end of G1 & help trigger progression through start transition
-Levels decrease in S phase
What do S cyclins: Cyclin A do?
-Forms complex with Cdk1 & Cdk2
-Bind Cdks after progression through start transition & helps stimulate chromosome duplication during S phase
-Levels remain elevated until mitosis; contributes to control of some early mitotic events
What do M cyclins: Cyclin B do?
-Forms complex with Cdk1
-Bind Cdk’s to stimulate entry into mitosis at the G2/M transition
-Levels decrease in mid-mitosis
How do cyclin Cdk complexes work?
Cyclins function by activating the Cdk (cyclin activating kinase; CAK needed for full activation)
-Cyclin protein also directs it to its specific target
Progression through which check point is different from the rest?
Metaphase to Anaphase
Metaphase to anaphase checkpoint is regulated via what? What complex is needed?
Proteolysis; anaphase promoting complex (APC/C; aka cyclosome)
What family is the APC/C; aka cyclosome part of?
ubiquitin ligase
APC/C; aka cyclosome is used to stimulate what? and what are the target proteins?
-Stimulates proteolytic destruction of specific regulatory proteins. (APC/C polyubiquitinated specific target proteins for destruction in proteasomes)
Target proteins: Securin, M-cyclins, S-cyclins
What are the early response genes?
usually, transcription factors-will induce the transcription of delayed response genes.
What are delayed response genes?
usually Cdks, cyclins, or other proteins needed for cell division
Growth factors bind to specific receptors to stimulate cellular growth & proliferation (turns on early response and delayed response genes)
Which phase of the cell cycle does this process belong to?
G1
In the G1 phase, what happens in response to the binding of the growth factor?
Cyclin D & E are transcribed & translated
-Cyclin D forms complexes with Cdk4 & 6(G1 Cdk complex)
-Cyclin E forms complexes with Cdk 2 (G1/S cdk complex)
Active G1-Cdk & G1/S-cdk allow for progression through the checkpoint
Active G1-cdk (and G1/S-cdk) complex will target a protein called______________.
RB
What does RB function as and which checkpoint is it associated with?
transcription co-repressor; G1/S checkpoint
What will inactivate RB?
Hyperphosphorylation
What does an inactive RB release to allow transcription to proceed?
E2F
What checkpoint phase is this diagram showing?
G1 phase->G1/S checkpoint
What 2 complex’s get targeted for destruction in the S phase?
cyclin D (G1-cdk complex) , and E (G1/S-cdk complex)
-promotes progression through the S phase
Active S-cdk complex allows progression through the S phase.
1) What was the S-cdk complex?
2) What is occurring during the S phase of the cell cycle?
1) Cyclin A: Forms complex with Cdk1 & Cdk2
2) DNA replication happens here
Phase G2:
s-cdk complex levels are _______
M-cyclin levels _____________
-still high
-begin to rise
M cyclin forms M-cdk, which cyclins and cdks are part of this complex?
Cyclin B; Cdk1
M-cdk complex is needed for what?
To pass through the G2/M checkpoint
-Bind Cdk’s to stimulate entry into mitosis at the G2/M transition
What happens to the S cyclins at the end of G2?
They are destroyed. Targeted for proteolysis by APC/C.
Once the M-Cdk complex is assembled, what happens to it?
It is immediately inhibited via phosphorylation.
Inhibitory phosphorylation in the M-phase occurs by a kinase called what?
Wee1
When the cell cycle is ready for mitosis to begin, the _____________ complex is ____________________
M-Cdk; de-phosphorylated
De-phosphorylation occurs by a phosphatase called what?
Cdc25
Cell progresses through the G2/M checkpoint and __________ begins.
Mitosis
What is needed for activation of various proteins needed for mitosis?
M-Cdk complex
An inhibitory protein that protects protein linkages that hold sister-chromatid pairs together in early mitosis.
Securin
Destruction of Securin activates a protease that separates ____________ allowing progression to ______________
The sister chromatids; anaphase
APC/C complex targets what? During what?
Securin; Metaphase to anaphase checkpoint in mitosis
What process is used to progress through M-to-A checkpoint?
Regulated proteolysis
After anaphase, the cell cycle continues through what?
Telophase -> cytokinesis
At the end of mitosis, the ___________ re also targeted for destruction by _________.
M-cyclins; APC/C
What happens after M-cyclins are destroyed?
-Inactivates most Cdks in cell
-Then many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases in the anaphase cell.
_______________________ is required for the completion of M phase, including the __________ & then _________.
Dephosphorylation; telophase; cytokinesis
Progression through G1 is delayed if:
-DNA is damaged by radiation, chemicals, or errors
-Absence of nutrients or growth factors
-Abnormal cell size
Entry of M is prevented when:
-DNA replication is not complete
-Presence of DNA damage
-Abnormal cell size
Progression of M-to-A is prevented if:
-Chromosomes are not properly attached to mitotic spindle
Binding of ____________ inactivates cyclin-Cdk complex.
Cdk inhibitory protein (CKI)
-Primarily used in cells to govern the activities of G1/S and S-Cdks early in cell cycle
What are the three important CKI’s?
p16, p21, p27
p16 inhibits:
Cyclin D-cdk4 & Cyclin D-cdk5 (G1-cdk complex)
p21 inhibits:
Cyclin E-cdk2 (G1/S-cdk complex)
Cyclin A-cdk2 & Cyclin A-cdk1 (S-cdk complex)
Cyclin B-cdk1 (M-cdk complex)
p27 inhibits:
Cyclin A-cdk2 & Cyclin A-cdk1 (S-cdk complex)
Cyclin E-cdk2 (G1/S-cdk complex)
Cyclin B-cdk1 (M-cdk complex)
What are the two tumor suppressor genes?
p53 & RB
p53:
-Recognizes and binds damaged DNA
-Unstressed cells have lower levels of p53 since it will be bound by a protein called Mdm2 & be degraded
RB:
-Generally found in active form (hypo or hyper phosphorylation)
-Can also recognize damaged cells
In the presence of DNA damage, p53 will be __________________, releasing ____________
Phosphorylated;Mdm2
(will not be degraded)
Active p53 binds DNA & promotes the transcription of ______
p21
p21 binds with _________, _________ it.
G1/S-cdk complex; inhibiting
An inactive G1/S-cdk complex will pause the cell cycle at the ____________ transition.
G1-S
RB: In the presence of a growth suppressor signal or DNA damage:
-p16 is transcribed; Inhibits the G1/cdk complex(needed to inactivate RB)
-RB remains activated & bound to E2F (no transcription of G1/S-cyclins or S cyclins-Cell is paused at the start of the transition)
Contact inhibition: The cell cycle progression can also be inhibited due to contact with:
-Other cells (density-dependent inhibition)
-A basement membrane or other matrix component (anchorage-dependent/most animal cells must be attached to a substratum in order to divide
How is contact inhibition reglated?
With cadherins & beta-catenin pathway
__________ promotes the cell cycle and prevent apoptosis.
Survival signals (PI3K-Akt-mTOR C Pathway)
Akt can promote the cell cycle progression by:
Activates/increases:
-Cyclin A->activation of CDK-1
-Cyclin D->activation of CDK-4/6
Decreases/inactivates:
p21 & p27
DNA replication occurs in what phase of the cell cycle?
S-phase
_____________ acts as a template for it’s own duplication.
DNA double helix
Each daughter cell will inherit a DNA double helix containing _______________________________
1 original strand & 1 new strand
(semi-conservative replication)
What are the steps of DNA synthesis?
1) Strand separation
2) Primer creation
3) DNA replication
4) Primer removal
What two proteins are needed to open up the DNA strand during the 1st step of DNA synthesis?
1) DNA helicase
2) Single strand binding proteins
DNA helicase:
-Unwinds double helix
What type of bond is DNA helicase breaking?
Hydrogen bonds
Single strand binding proteins:
Bind tightly and cooperatively to stabilize the single strand conforming
Opening of the double helix creates a ________________
Replication fork
At the replication fork a multienzyme complex, ____________________ will be used to synthesize both new daughter strands of DNA.
DNA Polymerase; can only add nucleotides to existing strand of DNA (works in 5’-3’ direction)
In what step of DNA synthesis does the replication fork appear?
1st step: Strand seperation
Step 2 of DNA synthesis:
The primer is made of _____ by an enzyme called ___________
RNA; DNA primase
Leading strand:
Synthesized continuously
Lagging strand:
Synthesized discontinuously
-The direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth
On the leading strand, how many primers are needed?
1
On the lagging strand, how many primers are needed?
1 primer is needed for each Okazaki fragment
How many nucleotides are there per each Okazaki fragment?
10 nucleotides long & made at intervals of 100-200 nucleotides on the lagging strand
Step 3 of DNA synthesis:
Once primers are built, _________________ can add nucleotides in the 5’-3’ direction.
DNA polymerase
Step 4 of DNA synthesis:
_________ are removed by a DNA __________ and replaced with ___________
RNA primers; repair system (RNAse H); DNA
Step 4 of DNA synthesis:
____________ joins 3’ end of the new DNA fragment with the 5’ end of the previous fragment.
DNA ligase
As the replication fork moves along the double-strand DNA, anything in front of the replication fork will become overwound forming ____________________
Supercoils
________________ relieves the super-helical tension by breaking the phosphodiester bond.
DNA topoisomerase
(this allows the two sections to rotate freely and relieve tension)
-The phosphodiester bond will reform as DNA topoisomerase leaves.
Label the diagram with the enzymes or proteins needed for each step:
Answers:
What must also be synthesized so that the newly replicated DNA can be packaged into nucleosomes?
Histones
Histone synthesis occurs where in the cell cycle?
S phase
Assist formation of assembly of histone octomer & nucleosomes:
Histone Chaperones (aka; Chromatin assembly factors)
How many mistakes occur for every 10^10 nucleotides during DNA replication?
1 mistake
DNA polymerase proofreading consists of what steps?
1) DNA polymerase activity
2) Exonucleolytic proofreading
3) Strand-directed mismatch repair system
DNA polymerase activity takes place when?
Prior to a new nucleotide being covalently added to the growing daughter chain.
Exonucleolytic proofreading occurs when?
Immediately after an incorrect nucleotide has been covalently added to the growing daughter chain.
Incorrectly added nucleotides will not provide an effective _________ end for ___________ to add on the next nucleotide.
3’-OH; DNA polymerase
A ________________ on DNA polymerase will initiate DNA polymerase to move in 3’-> 5’ direction, clipping off any unpaired residues.
Separate catalytic site
(3’-5’ proofreading exonuclease)
Since DNA replication occurs discontinuously on the lagging strand we end up with a shorter DNA fragment on the _______________ once the RNA primer has been removed.
daughter strand
Specialized nucleotide sequences at the end of their chromosomes:
Telomeres
(tandem repeats of GGGTTA)
-1 Telomere is 1000 GGGTTA repeats-
What recognizes the telomere DNA sequence?
Telomerase
Telomerase:
-Replenishes the sequence each time the cell divides
-The activity of telomerase varies based on the cell type
What type of cells have full telomerase activity?
Stem cells
Most cells have what type of telomerase activity?
Minimal
Telomerase gradually shorten untila descendant cell inherits chromosomes that lack telomere function. This initiates a response causing them to withdraw permanently from the cell cycle and cease dividing. This is called what?
Replicative Cell Senescence
Telomerase will recognize what?
The tip of an existing telomere DNA repeat on the parent strand & elongate it in a 5’-3’ direction.
(uses intrinsic RNA template as a template)
Following telomerase recognizing the tip of an existing telomere, what happens next?
The replication of the daughter strand can be completed by the conventional DNA polymerase.
(The extended telomere will be used as a template for synthesis of the daughter strand)
hat is the end product of mitosis?
2 identical daughter cells
What happens during prophase?
-Chromosomes condense (2 closely-associated sister chromatid)
-Mitotic spindles assemble between the two centromeres (centromeres have been replicated and are being moved apart)
What is a centrosome?
Protein organelle; consists of a pair of centrioles
What are centrioles surrounded by?
A cloud of amorphous material (pericentriolar matrix)
The minus ends of microtubules are associated with:
The centrosome
The plus end of microtubules do what:
Radiates out
Centrosomes undergo _____________ during the cell cycle in preparation for ___________
replication; Mitosis
What happens during prometaphase?
-Nuclear envelope breaks down
-Chromosomes attach to spindle microtubules via a protein called Kinetochore
Chromosomes attach to spindle microtubules via a protein called what?
Kinetochore
What happens during metaphase?
-Chromosomes align at the equator of the cell
(kinetochore microtubules attach sister chromatids to opposite poles of the spindle)
What happens during anaphase?
-Chromatids synchronously separate forming two daughter chromosomes
-Kinetochore microtubules get shorter while the spindle pole moves apart
(both contribute to the separation of chromosomes)
What happens during telophase and cytokinesis?
-Daughter chromosomes arrive at poles of the spindle
-Chromosomes decondense and a new nuclear envelope reassembles around each set
-Cytokinesis: cytoplasm divides in two forming daughter cells
What is the number of chromosomes in G1 phase of the cell cycle?
2n
What is the number if chromosomes in Prophase of mitosis?
2n
What is the number of chromosomes after cytokinesis?
4n
