PRIVETTE-VINNEDGE 2 Flashcards
What are the three phases of preparation for cell division in the cell cycle?
Interphase (G1, S, G2)
What are the growth phases of the cell cycle?
G1 and G2
What is the name of the DNA synthesis phase of the cell cycle?
S phase
What happens during the M phase of the cell cycle?
Mitosis (cell division)
How are transitions between the phases of the cell cycle regulated?
By cell cycle checkpoints
Where does DNA replication begin?
At distinct loci called Origins of Replication
What type of organisms have origins of replication with a consensus DNA sequence?
Prokaryotic organisms and yeasts
How do eukaryotic organisms identify origins of replication?
Based on contextual clues
What are three contextual cues that eukaryotic organisms use to identify origins of replication?
Local DNA topology; Chromatin modifications; DNA composition
What type of DNA sequence is usually found at origins of replication?
AT-rich sequence
What is the term for loosely packed chromatin that is open for transcription?
Euchromatin
What is the term for densely packed chromatin?
Heterochromatin
What type of chromatin is generally thought to be replicated first?
Euchromatin (early replication)
What type of chromatin is thought to be replicated later?
Heterochromatin (late replication)
What proteins bind to DNA origins of replication?
Origin recognition complex (ORC) proteins
What happens to many potential origins of replication in G1?
They are identified and primed, but only some “fire” and are used in S phase
How long does the S phase typically last?
8-10 hours
What proteins are involved in the E2F mediated transcription that occurs during the G1-S phase transition?
Cyclin-E and -A
What do Cyclin-E and -A activate?
CDK2
What does the CyclinE-CDK2 complex do to trigger the S phase?
It phosphorylates (inactivates) Rb
What roles do Cyclin E, Cyclin A, and CDK2 play in the S phase?
They have essential roles in initiating DNA replication
What is DNA origin licensing?
The process of identifying and preparing origins of replication for DNA synthesis
How many origins of replication are there per Mb of DNA?
25-40
What proteins bind to ORC during DNA origin licensing?
Cdc6 and Cdt1
What proteins are recruited after Cdc6 and Cdt1 bind?
MCM2-7 proteins
What is the result of inactivating licensing?
The pre-initiation complex
What phosphorylates Cdc6?
S phase CyclinA/E-CDK2 complexes
What protein binds to and inhibits Cdt1?
Geminin
What is the main purpose of licensing?
To prevent re-replication, ensuring DNA is only replicated once per cell cycle
What does DDK stand for?
Dbf4-dependent kinase
What does the DDK complex consist of?
Cdc7 and Dbf4
What does the DDK complex phosphorylate?
The Mcm complex
What does the DDK complex promote the loading of?
Cdc45 and Sld3
After cyclinA/E-CDK2 initiates licensing inactivation, what activates the MCM helicases?
GINS complex and DNA polymerase epsilon (pol ε)
What is the composition of the GINS complex?
A tetramer of Sld5, Psf1, Psf2, and Psf3
What is another name for the CMG complex?
Replicative helicase
What is the composition of the CMG complex?
Cdc45, MCM2-7, GINS
What is recruited to complete the replication progression complex (RPC)?
DNA polymerases
What does the formation of the replication fork allow?
DNA synthesis to begin
What makes up the replisome?
RPC + polymerases + PCNA + Replication Factor C
What is another name for Replication Factor C?
Clamp loader
What does PCNA stand for?
Proliferating Cell Nuclear Antigen
Describe the shape of PCNA.
Ring-shaped
What does PCNA encircle?
DNA
What is the function of PCNA?
Acts as a “sliding clamp”
What DNA polymerase does PCNA act as scaffolding for?
DNA polymerase delta (pol δ)
What is PCNA used as a marker of?
S phase
In what direction are both strands of DNA copied during replication?
5’ to 3’
What are the names of the two strands of DNA being copied during replication?
Leading strand and lagging strand
What proteins run ahead of the helicases during replication?
Topoisomerases
What do topoisomerases do to DNA?
Nick DNA to relieve tension from supercoiling and help unwind DNA
What is the result of the uncoupling of polymerase and helicase during replication?
Exposed single-stranded DNA (ssDNA)
What proteins coat the ssDNA when the polymerase and helicase uncouple?
RPA, then Rad51
What do RPA and Rad51 initiate after coating the ssDNA?
Fork reversal
How are stalled forks restarted?
Homologous recombination DNA repair or branch migration
What two proteins coordinate to control DNA replication and prevent re-replication?
Cdt1 and Geminin
Through what mechanism do Cdt1 and Geminin prevent re-replication?
DNA origin licensing
What can happen if Cdt1 accumulates incorrectly or geminin is lost?
DNA replication can occur multiple times without mitosis
What does FUCCI stand for?
Fluorescent Ubiquitination-based Cell Cycle Indicator
What does FUCCI allow for?
Live cell imaging of the cell cycle
What color is the Cdt1 protein fragment in FUCCI?
Kusabira-Orange 2 (mKO2)
What phase of the cell cycle does the Cdt1 protein fragment represent in FUCCI?
G1 phase
What color is the geminin protein fragment in FUCCI?
Azami-Green 1 (mAG1)
What phases of the cell cycle does the geminin protein fragment represent in FUCCI?
S-G2-M phases
Why were eGFP and RFP not suitable for FUCCI?
They were too stable, resulting in constant expression across the cell cycle.
Who developed FUCCI?
Asako Sakaue-Sawano
When and where was the development of FUCCI published?
In Cell in 2008
Why were Cdt1 and geminin protein fragments used in FUCCI?
Because they are only present during specific phases of the cell cycle and are rapidly degraded, providing accurate cell cycle phase information.
Why did the protein fragments in FUCCI have to be used together?
To distinguish between G1 and S/G2/M phases, as each fragment represents a different stage of the cell cycle.
In flow cytometry, what is used to label newly synthesized DNA?
Nucleotide analogs of thymidine, such as BrdU and EdU
What is the difference between BrdU and EdU detection?
BrdU uses antibodies for recognition, while EdU uses Click chemistry
What fluorescent dye binds to all DNA in flow cytometry?
7-AAD
What are CldU and IdU?
Thymidine analogs used to label newly synthesized DNA in DNA fiber assays
How are DNA fibers spread for analysis in DNA fiber assays?
On tilted slides
What method is used to label CldU and IdU in DNA fiber assays?
Immunofluorescence
What can DNA fiber assays be combined with to test their impact on DNA replication?
Various DNA damaging agents or gene expression variants
What can be rearranged in DNA fiber assays to test different hypotheses?
Order of pulses and treatments
What are telomeres?
Repetitive DNA sequences at the end of chromosomes
What happens to telomeres with each cell division?
They shorten due to incomplete replication of the ends of DNA (end replication problem)
Why are telomeres difficult to replicate during S phase?
Repetitive sequences confuse the polymerases, they form secondary structures (t-loop), and they are heterochromatic.
What is the t-loop?
A secondary structure formed by invasion of the 3’ end of the telomere into the duplex sequence with TRF1/2 proteins
What does the t-loop protect DNA from?
Degradation and being treated as a DNA double-strand break
True or False: You only want to replicate DNA once per cycle.
True, doing more replication will cause polyploidy (extra replicates of the chromosome(s)), or cancer
What is the Hayflick limit?
The number of times a cell can divide before it stops due to telomere shortening
What is the vertebrate telomere sequence?
5’-TTAGGG-3’
What is the full name of TERT?
Telomerase reverse transcriptase
What does TERT do?
Creates ssDNA from an RNA template
What is another name for the telomerase RNA component?
TR/TERC
True or False: PCNA can be an indicator of S phase in immunofluorescence.
True
Who won the 2009 Nobel Prize for the discovery of telomerase?
Carol Greider, Elizabeth Blackburn, and Jack Szostak
What does telomerase add to the chromosome?
DNA to the 3’ end
What DNA polymerase converts most of the ssDNA to dsDNA after telomerase extension?
DNA polymerase alpha/primase
What does TRAP stand for?
Telomere Repeat Amplification Protocol
What does TRAP involve?
In vitro creation of ssDNA by telomerase and PCR-based detection of telomeres by filling in the ssDNA
What is the typical appearance of TRAP reaction products?
A ladder of bands in 6bp increments (telomere repeat length)
What can loss of telomere capping and function cause?
Substantial genome instability
What happens to telomeres to protect the DNA when capping and function are lost?
They fuse
What does persistent telomere shortening result in?
Hayflick limit and senescence/apoptosis
In what percentage of tumors is telomerase upregulation found?
85-95%
Can hTERT overexpression alone immortalize normal somatic cells?
Yes, but it does not transform them
Give an example of a telomerase inhibitor that may be an anti-cancer drug.
Imetelstat
What are cohesins?
Protein complexes that promote sister chromatid cohesion after DNA replication
