29-DNA REPLICATION – Dr. Block Flashcards
What does semiconservative replication mean?
Semiconservative replication involves a conservation of one strand of each parent in each of the daughter DNA molecules.
What three observations have been made for all DNA polymerases?
All DNA polymerases share 3 common features:
- Inoming nucleotides selected by Complimentary base pairing with template strand
- New chain growth occurs in the antiparallel direction, 5’ to 3’
- All DNA polymerases require a primer strand to initiate synthesis
What is the function of DNA polymerase I? What is the Klenow fragment?
DNA polymerase I has three major activities:
- 5’ to 3’ polymerase: extends the length of the new DNA
- 3’ to 5’ exonuclease: proofreading activity
- 5’ to 3’ exonuclease: “snowplow effect”; any mispaired bases in front of the advancing polymerase are removed
The Klenow fragment is the larger fragment of DNA polymerase I that has the 5’ to 3’ polymerase and the 3’ to 5’ proofreading exonuclease.
What is the function of DNA polymerase III?
DNA polymerase III is the chief DNA replicating enzyme for E. coli. It cannot synthesis on intact duplex DNA, so it can only bind and polymerize SINGLE strand DNA at very high processivity. It also lacks repair mechanisms.
What is the function of a topoisomerase? A Helicase?
Topoisomerases (or “DNA Gyrases”) change DNA topology by cutting, adjusting the amount of supercoiling to be adjusted, and then religating the strands. Topoisomerase II introduces negative supercoiling.
Helicases on the other hand is responsible for unwinding the DNA double strand by disrupting the hydrogen bonding of base pair, while single strand binding proteins (SSB) keep the strands apart.
In replication, which strand is the leading strand and which is the lagging strand? Which is synthesized continuously, and which is not?
DNA polymerase can only synthesize in the 5’ to 3’ direction. When the helix is unwound the strand running in the 5’ to 3’ direction is the leading strand (template is 3’ to 5’), synthesized continuously with no breaks. The other strand is the lagging strand, running in the 3’ to 5’ direction (template is 5’ to 3’) is used discontinuously.
How is RNA used in DNA replication in prokaryotic DNA replication? What are Okazaki fragments?
Okazaki fragments are small DNA fragments that are synthesized from the lagging strand. These discontinuous fragments have RNA added to them to act as primer sequences for DNA Polymerase I. The polymerase binds and removes the ribonucleotides via 5’ to 3’ exonuclease and then uses the 5’ to 3’ polymerase to fill in with deoxyribonucleotides.
Where is E. coli DNA replication initiated and where is it terminated?
DNA replication for E. coli is initiated at a site on the strand called OriC.
Termination of replication occur at the binding of Ter to the Tau or Ter site by contrahelicase activity.
What are the processivities of DNA polymerases I and III?
DNA Polymerase I - processivity of 20 bases
DNA polymerase III - very high, possible processivity of 5 million
