Lec 3. DNA Replication Flashcards
What happened during 1953?
Watson and Crick describe the structure of the double helix
What happened during 1956?
Kornberg discovered DNA polymerase
What happened during 1958?
Meselson and Stahl demonstrate how the DNA template is used
After the structure of DNA was solved, what were 3 possible models for DNA replication proposed?
Conservative, Semi-conservative and Dispersive.
Define the conservative model for DNA
Conserving the original strand
Define the semi-conservative model for DNA
1 strand of old and 1 of new strand
Define the dispersive model for DNA
Mix of old and new strands
How did Meselson and Stahl demonstrate which model for DNA replication was used.
They created an experiment. Big picture: 2 different isotopes of Nitrogen, we label nitrogenous bases with DNA. We have the heavy isotope N15, N14 is the lighter isotope and will be higher. They grew bac in heavy N N15. Bac can only use N15 which makes only ds Dna with N15. We switch bacteria that can only grow in N14. we will see what happens when they grow. If the two old strands come back together. We will keep everything. Dispersive and semi= half and half and tied to double helix and density will be somewhere in the middle. Cant tell from the two. With semi conservative: we have half n half, but now since we are continuing this, the old strands can pair with the new and thus making new dna with the N14 light isotope.
Results: At the beginning we saw the heavy isotope. After 1 gen the band has shifted to the middle. After 2 rounds of replication where we see some intermediate density and some using only the light isotope.
Thus semi conservative replication for DNA.
Why was Kornberg confused?
He found an enzyme (DNA pol) that could replicate DNA but was not about to separate two DNA strands, only able to elongate from something else and could only go one direction down the dna strand
What are the three parts for DNA replication?
initiation, elongation, and termination
What are the steps in initiation?
Licensing and firing
Where does licensing occur?
G1
Where does firing occur?
S
What happens during licensing?
Getting ready to replicate. Origin of replication is located. Formation of pre-RC
What happens during firing?
We are ready to replicate, launching off to elongation. Formation of pre-IC
What does the pre-RC consist of?
ORC and MCM complex
What does the ORC do?
Binds to chromatin and designates that location as an origin of replication
How many origins do Prokaryotes have?
One per genome
How many origins do Eukaryotes have?
Many per genome
How do we find the origin in Prokaryotes?
AT-Rich bonds, DNA 1 prime sequence.
How do we find the origins in Eukaryotes?
CpG islands maybe G-quadruplexes, DNA 2 prime structure.
What is the MCM and what does it do?
a hexamer that forms the core of the pre-RC complex.
True or False? Only one MCM complex is needed at the first origin
False. Replication forks are bidirectional, a second MCM complex must be loaded.
What two important components distinguish the pre-IC from the pre-RC?
Functional helicase complex and presence of the DNA polymerase.
What is the functional helicase complex during firing called?
CMG
What are the three parts to the CMG?
Cdc45, McM2-7 hexamer, and GINS tetramer
How is the CMG helicase formed?
MCM, load on CdC protein that needs help with a kinase DDK, then GINS assisted by CDK kinase.
What factors are also involved in activating the CMG helicase complex?
Loading on DNA pol ε, Initial melting of the DNA duplex forming the replication bubble. Reconfiguring each helicase to associate with a ssDNA (this strand will become the leading strand).
Where are nucleotides added during elongation?
3’ end of the growing chain
In what direction does the chain grow in?
5’ to 3’ direction
During elongation, what are the 4 major steps to synthesize a new strand of DNA?
Add RNA primers, ADD dNTPs, remove RNA primers and seal gaps
What does primase do?
Add RNA primers
What does DNA polymerase do?
Add dNTPs
What does RNaseH do?
Remove RNA primers
What does DNA ligase do?
Seal gaps
True or False? Leading strand is synthesized continuously
True
What is the lagging strand made up of?
Okazaki fragments
What is the role of Helicase?
Unwinds DNA template
What is the role of Primase?
Synthesizes RNA primer
What is the role of RPC (replication protein C)?
Clamp loader, adds PCNA.
What is the role of PCNA (Proliferating Cell Nuclear Antigen)?
Sliding clamp, a processivity factor. Key pol associated with loading strand
What is the role of RPA (replication protein A)?
ssDNA binding protein, holds open replication fork
What is the role of DNA pol ε?
Leading strand synthesis
What is the role of DNA pol δ?
Lagging strand synthesis
What is the role of DNA pol α?
Adds initial dNTPs onto RNA primer
During elongation what is the rate of replication for prokaryotes?
1000 nt/sec
During elongation what is the rate of replication for Eukaryotes?
50-100 nt/sec
During elongation what is the total # of polymerases in prokaryotes?
5
During elongation what is the total # of polymerases in Eukaryotes?
14
During elongation what is the primase partner in prokaryotes?
Helicase
During elongation what is the primase partner in Eukaryotes?
Polymerase α
During elongation what is the sliding clamp for prokaryotes?
sliding clamp
During elongation what is the sliding clamp for Eukaryotes?
PCNA
During elongation what is the clamp loader for prokaryotes?
Clamp loader
During elongation what is the clamp loader for eukaryotes?
RPC (replication protein C)
During elongation what is the elongating polymerase for prokaryotes?
DNA pol III
During elongation what is the elongating polymerase for eukaryotes?
DNA Pol ε (leading) DNA Pol δ (lagging)
During elongation what is the ssDNA binding protein for prokaryotes?
ssDNA binding protein
During elongation what is the ssDNA binding protein for eukaryotes?
RPA (Replication protein A)
During elongation what replaces RNA primers in prokaryotes?
DNA pol I
During elongation what replaces RNA primers in eukaryotes?
DNA pol δ
During elongation what are the steps for the removal of the RNA primers?
Pol δ displaces the RNA primer, (continuing DNA synthesis until next okazaki fragment), FEN1 clips off the RNA tail, DNA ligase seals the gap.
During Elongation what does topoisomerase do?
Relieves stress on the helicase.
What is type 1 topoisomerase used in and how many gaps are made?
During replication and makes one gap on single bonds
What is type 2 topoisomerase used in and how many gaps are made?
During termination,two.
During termination what happens in the middle of chromosomes?
Superhelical stress increases and daughter helices intertwine, leading strands pass each other only to stop when an okazaki fragment is encountered, RNA primers are removed, MCM complex is polyubiquitinated thus signaling for its degradation, topo II removal of daughter helix entanglements.
