Flashcards in Molecular Biology Lecture 7 - DNA Replication Deck (23):
DNA synthesized in the ________ direction by ________
5’ → 3’ , DNA polymerase
Proofreading is done by ______ in the _______ direction
exonuclease, 3’ → 5’
What are nucleoside triphosphates (dNTPs)?
Nucleoside triphosphate (NTP) is a molecule containing a nucleoside bound to three phosphates. These include the four bases needed for DNA replication.
A particular arrangement of single-stranded DNA (ssDNA)
Correct base pairing provides specificity
3’ OH and α Phosphate correctly positioned.
Right hand glove model for DNA pol
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DNA polymerase form processive reaction. This means?
Processivity is a characteristic of enzymes that operate on polymeric substrates. In the case of DNA processivity is defined as the average number of nucleotides added each time the enzyme binds a primer:template juction
1. Binding of pol to DNA is the rate-limiting step
2. Polymerization is very fast
3. Processive – multiple nucleotide additions per polymerase binding
Proofreading of DNA....
....is done by the exonuclease in the 3' to 5' direction. Mispaired DNA alters the geometry of the 3'-OH. This will decrease the rate of addition and increase the rat of exonucleases
1. Unpaired (SS) DNA slows down the polymerase
2. Exonuclease removes mismatched base
3. 10-5 vs. 10-7 errors
4. Post-replication repair reduces errors
DNA synthesis on two strands
Two DNA polymerase molecules
Leading and lagging strands - Okazaki fragments
Primase are short RNA primers (5-10 nucleotides long) on an ssDNA template.
DNA polymerase requires a primer to copy ssDNA, RNA primer can be created de novo (the method of creating a transcriptome without a reference genome)
RNA primer Con't.....
1. RNA not DNA primer
- 5-10 bases
- RNA polymerase
- Does not require a primer
3. Recognizes simple sequence (GTA in E. coli)
4. Stimulated when bound by helicase
- restricts activity to replication fork
Removal of primer
To RNA primers with DNA, an enzyme RNaseH recognizes and removes most of each RNA primer. RNaseH recognizes and degrades RNA base paired with DNA.
RNaseH can only cleave bonds between RNA:DNA
The removal of RNA leaves a gap in the dsDNA.
DNA polymerase I fills this up from the 5'-3' end
DNA ligase – covalent linkage, requires ATP
1. DNA polymerase does not unwind DNA efficiently
2. Helicase moves in front of polymerase
Moves along SS and denatures DS
3. Requires ATP
4. Hexameric protein - circles DNA
5. Polarity of movement
5’ → 3’ (lagging strand)
7. Requires DNA to be SS first (initiation).
After DNA helicase passes, the newly separated ssDNA must remain free for DNA polymerase to use a template for DNA synthesis.
ssDNA proteins (SSB's) rapidly bind to the separated strands
1. Cooperative binding
2. Cannot denature DNA
3. Stabilize ssDNA
4.Non-specific binding: electrostatic interactions
5. Easily displaced
As the strands of DNA are uncoiled by DNA Helicase on the replication fork it increasingly becomes super-coiled.
1. Topoisomerase II relieves supercoiling
2. DNA gyrase (+1 to -1 in figure)
3. Remember that the E. coli chromosome starts out negatively supercoiled (DNA gyrase).
Prokaryotes and eukaryotes have multiple DNA polymerases. In E.coli ......
1. E. coli
- DNA pol I – primer removal, gap filling
- accurate, not processive
- DNA pol III – main replication pol
- accurate, highly processive
- Other DNA pols involved in DNA repair
DNA polymerases in Eukaryotes
- DNA pol α/primase – primer synthesis, initial DNA synthesis
- DNA pol δ – lagging strand synthesis
- DNA pol ε – leading strand
- Other DNA pols involved in DNA repair or mit DNA replication
DNA polymerase switching
Overview of DNA replication
1. DNA synthesis
3. Primer removal
4. Filling in gap and covalently sealing
5. Producing SS template
6. Relieve positive supercoils
7. Processivity - sliding clamp and clamp loader
- initiation of replication
- cell-cycle regulation
These are proteins composed of multiple identical subunits that assemble in the shape of a donut.
in the absence of a replication fork, DNA Polymerase dissociates and diffuses away from the template DNA on average one every 20-100bp synthesized.
In the presence of the sliding clamp DNA polymerase may still disengage but will not diffuse away from the DNA
Sliding clamp mechanism
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