11.3 exam 2 slides Flashcards
What is the DNA Pol III core made up of?
Heterotrimer • 3 parts: • α = polymerase activity • ε = Proofreading (3’ 5’ exonuclease activity) • θ = unknown
What is the DNA Pol III holoenzyme made up of?
The entire Pol III enzyme
• 3 cores
• 3 sliding clamps
• 1 clamp loader (ATP dependent)
What is the β clamp?
- Allows for processive synthesis
* The Pol III core stays in the proximity of DNA
What is the clamp loader?
• Loads and unloads β clamps to DNA • ATP dependent activity 1. Opens β clamp 2. DNA binding of clamp loader • ATP hydrolysis causes dissociation of clamp loader
What does DNA helicase do?
NTP dependent (usually ATP)
• 5’ 3’
• (Opposite in eukaryotes!)
• Ring-shaped hexamers of DnaB
Does helicase improve in speed with the aid of Pol III?
Yes
What is the bps/s of helicase before and after Pol III?
- Helicase without Pol III = 35 bps/s
* Helicase with Pol III = 700 bp/s
Primase in prokaryotes:
- DnaG primase
- RNA primer
- Interacts with helicase
- Primer length: 11 to 13 nt
Pol I and ligase in prokaryotes:
Pol I • Previous section slides • Interacts with β sliding clamp Ligase • ATP dependent • Repairs covalent bond • Acts on 5’ DNA terminus • Interacts with β sliding clamp
RNAseH function:
- RNAseH can also assist in removing RNA
* Cannot remove the last nucleotide • Still needs the assistance of Pol I
Can RNAseH remove the last nucleotide?
No
What does SSBPs stand for?
single stranded binding proteins
What do SSBPs do?
- Prevent hydrogen bonds from reforming in separated DNA
- Prevent action of endonucleases from attacking opened DNA
- Remove hairpin structures
What does the replisome do?
- Replisome = Pol III holoenzyme + DnaB helicase + primase
* 700 bp/s
What does the trombone model explain?
Loop formation involves:
1) An RNA primer via primase
2) A clamp
Polymerization occurs until the start of a new Okazaki fragment.
Loops are traded between the 2 lagging strand polymerases
How many polymerases are involved in the trombone model?
3 polymerases
What is clamp recycling?
- β clamps are left behind on each completed Okazaki fragment
- DNA Pol III hops to the next β clamp
- Okazaki fragments to β clamp ratio is 10:1 Clamps must be recycled!
Do clamps have to be recycled?
YES
EUKARYOTES
Eukaryotes after this
What is the MCM in eukaryotes?
- Helicase is called MCM
- MCM requires the assembly of multiple proteins to become an “active helicase”
- All together, these proteins are called CMG
- Hexamer
What is helicase called in eukaryotes?
MCM
What does MCM require to become an active helicase?
multiple proteins
All together what is the name of the multiple proteins that the MCM requires to become an active helicase?
CMG
What are Eukaryotic polymerases?
- α = primase
* δ = chromosomal replication • ε = chromosomal replication
What is the CMG complex?
Opens 3’ 5’ • Opposite prokaryotes • Cdc45 • MCM (helicase) • GINS
What is DNA poly α?
- Primers of 25-40 nt
* No 3’5’ exonucleases activity (no proof reading)
What polymerases complete chromosomal replication in eukaryotes?
Chromosomal replication is completed by Pol δ and Pol ε
• δ lagging
ε leading
• Both have DNA polymerase and 3’5’ exonuclease activity
What is the eukaryotic replisome called?
The RPC
What does RPC stand for?
Replisome Progression Complex
What does replication protein a or (RPA) act like in eukaryotes?
The SSBps previously discussed with prokaryotes
clamp in eukaryotes:
- Sliding clamp = PCNA
- No sequence homology to β clamp • But nearly superimposable, cool!
- Clamp loader = RFC (Replication Factor C)
How are okazaki fragments removed in eukaryotes?
- RNA primer is “lifted” by the DNA Pol δ
* RNA primer is degraded by FEN1 and Dna2 5’ 3’ activity
Where does replication originate?
At an origin of replication
What is a replicon?
The total DNA replicated from 1 origin
Generally in bacteria 1 _____ and 1 ______ = entire chromosome
origin ; replicon
initiator protein in prokaryotes:
DnaA
Origin must be inactivated to prevent second round of _____ in prokaryotes?
replication
Initiation regulation in prokaryotes:
- HemimethylationatGATCattracts SeqA
- SeqA blocks DnaA
- DnaAhydrolyzesATPafter initiation
- Can bind DNA but unable to open it
- HdainsuresDNA-ATPhydrolysis
- WhenRNApolymeraseisabsent —the DNA doesn’t open as well
Eukaryotic initiation:
- Multiple origins of replication
- Each origin fires once
- Occurs in S phase
- Discrete origins have been identified in yeast, but not easily in other organisms
How many times do eukaryotic origins fire?
once
in eukaryotes, if you weren’t marked in ____ you won’t fire in ____
G1 ; S
When do prereplication complexes form in eukaryotes?
During S
Describe Eukaryotic initiation:
- G -phase 1
- ORC binds
- Licensing proteins attract the 2 MCM helicase • Cdt1
- CDC6
- Prereplication complex (preRC) = ORC + 2 x MCM + licensing proteins
- S-phase
- Kinases trigger replication through phosphorylation
How is replication triggered in the eukaryotic S phase?
Kinases trigger replication through phosphorylation
What does termination accomplish in bacteria?
prevent collisions of polymerases
What does termination accomplish in eukaryotes?
how do you replicate the ends of linear chromosomes
Describe E, coli termination sites:
- 23 bp sequences = termination sites
- 2 clusters in either direction
- Tus proteins bind Ter sites
- Tus-Ter complexes has polarity (2 directions)
- Nonpermissive direction = blocks replication
- Permissive = permits replication
What does non permissive direction do?
blocks replication
What does permissive direction do?
Permits replication
Where do Tus proteins bind?
Ter sites
E. coli Ter sites have how many clusters in either direction?
2
Do tus-ter complexes have polarity?
Yes, 2 directions
What are the 2 directions of tus-ter complexes?
- non permissive (block replication)
- permissive (permits replication)
Where are tus-ter complexes found?
E. coli
T/F Replication forks are not allowed to travel past her sites oriented in the opposite direction
True
What happens in equal fork speed in E. coli?
Replication forks meet in terminus region.
What happens in unequal fork speed in E. coli?
Clockwise fork blocked by her cluster
Describe E. Coli replication and transcription:
- usually in the same direction
- Prevents collision of DNA pol and RNA pol
- Codirectional collisions do not cause impedence
What is the end replication problem in linear chromosomes?
- After removing the primers on the ends of linear chromosomes there is a 3’ overhang
- That is, there is no free 3’ –OH group to finish the chromosome ends
- Telomeres shorten with every cell division
- Shorten may encroach in genic regions
What does telomerase do?
- Telomerase reverse transcriptase = creates DNA complete from RNA
- Telomerase RNA = RNA template
- Telomerase = TERT-TR holozenzyme
Even WITH telomerase, what will happen?
there is a still a 3’ overhang
What is the telomere capped with?
Shelterin
What happens to the overhang of the telomere?
It is looped back as a t-loop
Shelterin and t-loop prevents ________
- cell cycle arrest
- telomeres from joining other chromosomes
- overzealous telomerase activiity
What do shorter telomeres correlate with?
More age related diseases
Longer telomere are not necessarily correlated with longer life but ARE correlated with _____-
a healthier aging life
Adding telomerase to mouse models does what?
Shorten lifespan in terms of increases cancer-related deaths
Telomerase is extinguished during what?
Embryonic differentiation in most somatic cells—- remains active in few cell lines like germ cells
