MBG PART TWO: Lecture 2 Flashcards
What doe DNA replication require?
- DNA dependent DNA polymerase
- Four deoxyribonucleoside triphosphates (dNTPs)
- A single stranded template of DNA to be copied
- An RNA primer (provides the 3’-OH end to initiate DNA synthesis by DNA
polymerase)
What direction is DNA synthesized in?
5’ to 3’ direction
What are the characteristics of the newly synthesized DNA strand?
Complementary and Anti-parallel to parent strand.
What are DNA strands held together by?
Hydrogen bonds between complementary bases
What happens since DNA is antiparallel and synthesis proceeds in the 5’ to 3’ direction?
DNA replication must proceed n opposite directions on each strand.
What happens on the leading strand?
DNA synthesis is continuous (same direction as unwinding)
What happens on the lagging strand?
DNA Synthesis is discontinuous (opposite direction as unwinding)
What does the lagging strand create?
Okazaki fragments
What is the Eukaryote size of Okazaki fragments?
0.1 - 0.2kb
Where does all DNA replication start?
At an origin (Note the differences in origin between Theta, Rolling Stone and Linear Eukaryotic)
For prokaryotic DNA where does all replication begin at?
An “origin”
What is the replication region called for E.coli?
OriC
What is the Tandem 13-mer sequences?
13 base pairs that houses the AT-rich region
What is the Four 9-mer initiation protein binding sites?
The 4-DnaA binding sites
What is the function of the DnaA proteins?
They are initiator proteins that bind to oriC (replication origin) and cause a short stretch of DNA to unwind
What are the first three steps involved in Prokaryotic DNA replication?
1.) Initiator proteins (DnaA) bind to orC (origin of replication)
2.) They unwind a small strand of DNA
3.) The unwinding allows helices and Single Stranded Binding Proteins to attach to the single stranded DNA
Where does Helicase only bind to?
Single Stranded DNA (this is unique to prokaryotes) and disrupts the hydrogen bonding between complementary pairs
What is DNA helicase (prokaryote)?
Unwinds the DNA in the 5’ to 3’ direction: it travels only on the lagging strand ahead of the replication machinery.
What is Single-Strand Binding Proteins?
Protect and keep DNA single stranded (avoiding hairpin)
What is DNA gyrase?
A topoisomerase that makes double stranded breaks in the DNA to relieve the torsional strain that builds up from helicase.
What are the second three steps involved in prokaryotic DNA replication?
1.) DNA helicase binds to the lagging strand template at each replication fork and moves in the 5’ to 3’ direction, breaking hydrogen bonds and moving the replication fork.
2.) SSBP stabilize the exposed single stranded DNA
3.) DNA gyrase relieves the strain ahead of the replication fork.
What is Primase (RNA polymerase)?
Synthesizes a short RNA primer that provides the 3’ OH end for DNA polymerase to begin DNA synthesis
Where does primate bind to initiate synthesis?
Helicase
How does the replication fork proceed?
Bidirectionally
What is DNA polymerase?
Elongates the new nucleotide strand by catalyzing DNA polymerization. Requires a primer.
How many DNA polymerases are there in E.coli?
5
DNA Pol III
Main DNA polymerase. Has 5’ to 3’ polymerase activity and 3’ to 5’ exoncuclease activity.
It can add a dNTP on the 3’ end but can also back up to remove a nucleotide that has been mis-incorporated.
DNA Pol I
Similar to DNA Pol III but has 5’ to 3’ exonuclease activity
What does DNA Pol I 5’ to 3’ exonuclease activity mean?
It can remove the RNA primer and replace it with DNA
What happens at the leading strand?
DNA Pol III synthesizes continuously making a new strand
What happens at the lagging strand?
DNA synthesis by polymerase III occurs in “sections” - discontinuous
What happens to DNA Pol III on the lagging strand when it reaches the 5’ end?
It is swapped for DNA Pol I.
What does DNA ligase do?
Makes a phosphodiester bond between the 5’ phosphate and the 3’ OH group joining Okazaki fragments (replaces nicks)
E.coli polA mutants have a defective DNA polymerase that lacks 5’ to 3’ exonuclease activity. How will this affect replication?
DNA polymerase I will not be able to excise the RNA primers.
