Ch. 11 DNA Replication (part 1) Flashcards
How is DNA replicated? (2 descriptors)
semiconservatively and semidiscontinuously
Where does DNA replication begin? How does this differ between prokaryotes and eukaryotes?
Begins at an origin of replication (ori).
Prokaryotic circular chromosomes have one ori and two replication forks.
Eukaryotic linear chromosomes have multiple ori and multiple replication forks.
What does it mean that DNA replication is semidiscontinuously?
Leading strand is continuous
Lagging strand is discontinuous
What is DNA synthesis done by?
polymerases
How do DNA pol’s extend DNA?
ONLY extend DNA 5’ → 3’ (add to the 3’ end)
What do DNA pol’s require to synthesize DNA? (2)
They require a template strand to guide new strand synthesis through base pairing.
They require a primer containing a free 3’-OH group.
What is the primer for DNA pol in cells?
RNA (placed by RNA pol)
How does proper base pairing occur with DNA pol?
Only proper base pairs can fit into the active site of DNA pol. Incorrect bases are not in line.
(get an error every 10⁴ - 10⁵ bases)
How does a nucleotide get added to a growing DNA strand by DNA pol? (3)
- the 3’-OH of the primer attacks the ɑ phosphate of the dNTP
- pyrophosphate (PPᵢ) is released
- DNA pol slides one base forward and repeats
What does the action of DNA pol require as far as enzyme structure? (2)
Two active sites…
1. insertion site (new base)
2. postinsertion site (previous position)
What feature/activity do many DNA pol’s have?
3’→5’ exonuclease activity
What is a nuclease?
enzymes that can break phosphodiester bonds
What is an exonuclease?
a nuclease that shortens DNA from the ENDS
What is an endonuclease?
a nuclease that shortens/cuts DNA INTERNALLY
What does 3’→5’ exonuclease activity do?
It removes erroneous base pairs and increases accuracy by 10² - 10³. (DNA PROOFREADING)
Where does 3’→5’ exonuclease activity occur in the DNA pol?
At a different active site than synthesis.
What feature/activity do a few DNA pol’s have?
5’→3’ exonuclease activity
What does 5’→3’ exonuclease activity do? (3)
It removes RNA primers during replication and is involved in DNA recombination and repair.
How many DNA pol’s does E. coli have?
five
What does DNA pol I of E. coli do?
It removes RNA primers and is used during DNA repair mechanisms.
What does DNA pol II, IV, and V of E. coli do?
They are expressed following extensive DNA damage and are not very accurate. (NO 3’→5’ exonuclease activity)
What does DNA pol III of E. coli do?
It is the main DNA pol for chromosome replication.
What does a DNA pol structure resemble?
A right hand
What does the “palm” of DNA pol contain?
It is where DNA sits. The pol active site.
What do the “fingers” of DNA pol contain?
A dNTP binding site
When does the closed conformation of DNA pol form?
When a proper base pair is formed
What happens if an incorrect base pair forms in DNA pol?
The closed conformation is prevented and a slight conformation change allows the DNA to associate with the 3’→5’ exonuclease domain (to remove the incorrect base).
What “chemical” does DNA pol require?
divalent cations, mainly Mg²⁺
How do divalent cations associate with DNA pol?
conserved Asp residues (aa) bind two Mg²⁺ ions
What do the divalent cations do and how do they do it (2)?
They catalyze bond formation or breaking. (NO direct aa involvement, only the divalent ions!)
- one ion deprotonates the 3’OH to form a 3’O⁻ nucleophile
- the other ion binds the dNTP to facilitate PPᵢ removal
(similar mechanism for the 3’→5’ exonuclease)
What are the other proteins involved in DNA replication? (5)
DNA helicase, topoisomerase, SSB protein, primase, and DNA ligase
What is DNA helicase?
An enzyme that breaks H-bonds to separate parent strands using NTP hydrolysis as the energy source.
What is topoisomerase?
An enzyme that alleviates the supercoiling that separation of parent strands by DNA helicase causes.
What is SSB protein?
Single strand binding protein: binds ssDNA to protect it from nucleases and prevent re-annealing.
What is primase?
An enzyme that creates short RNA fragments to initiate DNA pol.
What happens to primers after DNA synthesis?
Primers are eventually removed after DNA synthesis by a DNA pol.
What is DNA ligase?
An enzyme that links Okazaki fragments together.
Does each replication enzyme work independently? Why or why not?
No. A complex holoenzyme is built for DNA replication. It allows for the simultaneous replication of leading and lagging strands.
What does the replication holoenzyme contain? (3)
three pol subunits (all pol III bcs. its bacteria)
three β sliding clamps
one clamp loader
What is the difference between DNA pol III on its own and the holoenzyme/complex?
On its own it can synthesize 10 nt/s. In the complex it can synthesize 1000 nt/s.
What is processivity?
It is the addition of subunits without dissociating from the template. It is also used as a measure of speed.
What is the purpose of β clamps?
They keep DNA pol in contact with DNA. When the pol dissociates, it will quickly rebind because the clamps keep it from floating away. (increase processivity)
What is the purpose of the clamp loader?
It attaches β clamps to the DNA. It requires ATP hydrolysis!
What is the replisome?
It contains all of the DNA replication proteins mentioned and can synthesize the leading and lagging strands simultaneously.
How many polymerase cores are on the leading strand? Lagging strand?
Leading strand has one polymerase core.
Lagging strand has two pol cores so it can keep up.
What does lagging strand synthesis create?
It creates DNA loops that are described by the trombone model.
How is eukaryotic replication generically described?
It is much like bacterial replication, but more complex and less understood.
How does eukaryotic replication differ from bacterial? (4)
1.Different pol’s are used for leading (ε) and lagging (𝛅) strands
2.Forks move slower than in bacteria and create shorter Okazaki fragments
3. Helicase moves 3’→5’
4. Removal of RNA primers is different
How many origins on bacterial chromosomes?
One ori
How is bacterial replication initiated generally?
DnaA binds to sites near A=T rich regions causing bending of the DNA that opens it and allows helicase to bind.
How is binding of DnaA regulated and why?
Binding is regulated by multiple Dam methylase sites (GAₘTC) to prevent newly synthesized daughter DNA from being immediately replicated.
How does Dam methylase regulate DnaA? (3 ish)
Daughter strand is NOT methylated but parent strand is.
1. the hemimethylated DNA attracts SeqA
2. SeqA blocks DnaA from binding the ori
3. A=T rich regions stay together, so no replication
Does the daughter strand stay unmethylated forever?
No. Eventually the daughter strand will be methylated.
What happens when the daughter strand is methylated? (2)
- SeqA dissociates from the fully methylated DNA
- DnaA can now bind and initiate replication
What else can affect the ability of DnaA to initiate replication, besides Dam methylase? (2)
- There are twice as many DnaA binding sites after replication and not enough copies of DnaA to initiate. (DnaA is also a regulatory protein)
- Open complex formation is ATP-dependent and the release of ADP accompanied by binding of new ATP can take 30 min. (“timer mechanism”)
How many origins do eukaryotic chromosomes have and why (2)?
Multiple origins because the chromosomes are larger and replication is slower.
How far apart are the origins of eukaryotic chromosomes?
~10﹣40 kb apart
Where do eukaryotic replication forks meet?
between ori sites
How and when is eukaryotic replication initiated?
In G1, a protein complex forms on the origins.
What is the complex that forms on euk. origins initiating replication?
Origin recognition complex (ORC): complex that is made of many proteins, binds to the ori, and is ATP-dependent.
What happens after the ORC binds at the ori? (2)
Additional proteins bind to the ORC and ori. These proteins attract other proteins associated with the replication complex.
How do eukaryotes regulate replication initiation?
Initiation proteins are INACTIVATED once replication begins to prevent initiation until the cell enters G1 again.
