Exam 2-3 Euk Flashcards
When does replication start in a eukaryotic cell?
During the S phase of the cell cycle.
Where can replication start for a eukaryote?
Initiation occurs at multiple origins.
What two events involves initiation of replication?
- Replicator Selection
2. Origin Activation.
Explain the replicator selection broadly.
The replicator selection is mediated by the origin recognition complex (ORC) during G1 phase.
A pre-replication complex is formed at the origin. This complex needs to be activated to allow replication to occur and this depends on the several kinases.
All of this occurs in the S phase.
What are cyclins?
Cyclins are proteins that are produced in cells that dictate what cell cycle phase a cell is in.
Depending on the type of cyclin at that specific cell cycle phase, different kinases called cyclin-dependent kinases are activated or inactivated.
What occurs in the G1 Phase of replication in eukaryotes?
You will have the association of Cdc6 and Cd1 (helicase loaders) and Mmc2-7 (helicase).
They will just bind to DNA forming a pre-replicated complex that will be ready to initiate once the cell hits S-phase.
Why S phase so important in replication?
Because the only time Cyclin-E is present is during the S-Phase.
Cyclin-E allows the activation of a cyclin-dependent kinase cdk-2.
What happens once Cyclin E is present?
Once you have cyclin-dependent kinase, it will form a complex with Cyclin E, activating the kinase.
In this case, it will phosphorylate the helicase loaders, and it will disassociate the loaders from the DNA.
Phosphorylation will also occur to the helicase as well and that will transition to get onto single stranded DNA, and that will really allows the opening up of the DNA.
What happens after your DNA has opened up after phosphorylation?
Next comes in a bunch of polymerases.
You have specialized polymerases that will do lagging strand or leading strand synthesis.
You have delta, and epsilon polymerases along with alpha polymerase with a primase will associate with the DNA strand.
After a primer is placed down, synthesis will occur.
What is unique about the binding between DNA and DNA helicase when comparing Eukaryotes to Prokaryotes?
In eukaryotes, the helicase binds to a double stranded DNA molecule.
In prokayotes, the helicase binds to a single-stranded DNA molecule.
The activation of the pre-replicative complex depends on what?
The two kinases.
What happens after a primer is laid down during eukaryotic replication?
So first, MCM (helicase) unzips the DNA and RPA (single-stranded binding proteins) binds to stabilize the single stranded DNA.
DNA Polymerase-alpha with a primase complex will come in and set down a primer.
After it has set down a primer, DNA Polymerase delta or epsilon will switch with DNA Polymerase-alpha and continue synthesis.
How many times will DNA replicate in a eukaryotic cell?
Only once at the most per cell cycle.
What are telomeres?
They are non-coding short, tandem repeats of DNA.
They serve as protective “caps” on the ends of chromosomes.
Which strand be synthesized all the way to the end? Which strand cannot?
The leading strand can replicate all the way to the end of the DNA strand.
The lagging strand will have a 3’ overhang. Even if a primer was placed there, there would still be an overhang, due to the fact that the primer would later have to be removed.
What enzyme solves the end replication problem?
Telomerase.
Describe the enzyme telomerase.
It is a ribonucleotide protein that contains the telomerase reverse transcriptase (TERT) and an RNA component.
Describe the mechanism of Telomerase.
The ends of chromosomes contain a G-rich area known as a telomere.
Telomerase recognizes the telomere and using an RNA template (primer) within the enzyme, telomerase elongates the strand in the 5’ -> 3’ direction and adds addition repeats to the parental strand as it extends the telomere.
The lagging strand is then synthesized by DNA polymerase-alpha which carries a DNA primase within its subunit. This way, the whole DNA sequence is copied onto the new DNA strand.
What is the purpose of a T-loop?
Telomeres swing back to form a loop because you do not want a single stranded DNA hanging out by itself. It would make it susceptible to nucleases. It protects the strand.
What stops the growth of the telomere end?
Telomere binding proteins regulate telomerase activity and telomere length. The binding proteins will add onto the DNA strand as a faster rate as compared to Telomerase until it “pushes” it off.
What is the role of a histone chaperone?
It take transports histones from the old DNA strand and breaks it apart for the new strand.
Why is it crucial that there are some old and some new histones on the parental and daughter strands?
Following DNA replication, there will not be enough histone tetramers to bind all of the newly synthesized DNA, so newly synthesized histones are needed to help with the coiling of all of the new DNA. The only problem is that the newly formed histone proteins do not have the modifications (methylation, acetylation, phosphorylation, or ubiquitylation) that help regulate the gene activity. When the nucleosomes of the two new strands are formed, they will contain a mixture of both the old and new histone tetramers (H3/H4) and dimers (H2A/H2B). This is important that both strands receive some of the old histones because they contain the epigenetic modifications mentioned earlier (methylation, acetylation, etc). Then there are proteins (bromodomain proteins) that associate with the DNA strands following replication and utilize the modifications on the old histone subunits to recruit other proteins to make the same modifications on the newly introduced histone proteins. This step is vital to maintain gene regulation and ensure that similar levels of the genes on the newly replicated strands will be expressed.
