DNA Replication Intro, Priming and Methods Flashcards
(28 cards)
What three things must DNA replication be?
high fidelity
highly processive
relatively fast
WHy cant DNA replication be too accurate?
No genetic change will occur ie mutations and natural selection
Why is DNA replication highly processive?
DNA Polymerase can bing and stay attached to the DNA and replicate it for long periods of time before becoming unstable. This allows large chucks of the genome to be replicated before the bond breaks
What is semiconservative replication?
DNA is typically a double stranded molecule which means that each of the two strands could possibly act as a template for its “partner” strand resulting in each daughter duplex containing a strand that is the same as one of the parental strands… one of the parental strands is “conserved”
What are the three ways in which DNA replication could proceed?
semiconservatively
conservatively
dispersively
What was the contribution by Melselson and Stahl?
They demonstrated that replication is a semiconservative process by employing CsCl gradient ultracentrifugation of DNA labeled with heavy Nitrogen (15N).
What was the experimental process completed by Melselson and Stahl to show that DNA replication is a semi conservative process?
They labeled DNA cells by growing them in HEAVY Nitrogen (15N). Then they moved the cells grown in the heavy Nitrogen to a medium that contained normal LIGHT Nitrogen (14N). Then they subjected the DNA to CsCl gradient ultracentrifugation to determine the density of the DNA. DNA migrated through the material and at the top heavy DNA is more dense and eventually mixes with the light to make it light and dark till it eventually becomes light. It concluded that DNA replication was a semi conservative process because half was dark and half was light and it eventually became light.
WHat other three possibilities could DNA replication be described as?
continuously
semi-discontinuously
discontinuously
What did Okazaki propose?
That DNA polymerase could make one strand of DNA CONTINUOUSLY in the 5’—>3’ direction but had to make the other strand discontinuously in the 3’—->5’ direction.
What is the continuously synthesized strand called?
the leading strand
What is the discontinuously synthesized strand called?
The lagging strand
What were Okazaki’s two experimentally testable predictions about replication? And how is each prediction tested?
- If short pieces of DNA synthesized on the lagging strand, then it should be possible to “catch” those short pieces of DNA before they are linked together. This is tested by employing short pulses of DNA radio labeling.
- If the enzyme DNA ligase is eliminated from the replication process, then the short pieces of DNA made ought to be detectable even at longer labeling periods. To test this hypothesis Okazaki use T4 phage mutants that were defective in the production of LIGASE.
What were the methods and results of Okazaki’s semi-discontinuous replication?
Phage T4 DNA was labeled with very short pulses of radioactive DNA and the DNA product was separated according to size by ultracentrifugation. With the presence of ligase short DNA pieces were only found in the beginning and then gradually were transformed into longer pieces. Without the presence of DNA ligase short DNA pieces accumulate and no long pieces were present even after a long period of time.
WHat direction is DNA replication proceed in?
It is replicated in the 5’—>3’ direction.
WHat is the priming of DNA synthesis?
The double stranded two parental strands separate. The to get DNA replication to commence, RNA primers must be laid down on the template strands to provide a double stranded region for DNA polymerase to bind and add on to.
What is a primer?
piece of nucleic acid (short piece of RNA) that the polymerase cab “grab on” to and extend by adding nucleotides to its 3’ end
DNA replication is ________ with DNA synthesis occurring simultaneously at 2 replication forks.
bidirectional
What is the oriC and what does it stand for?
It stand for origin of replication and it is a fixed starting point within the DNA were replication is initiated.
What was the experiment done by Cairns?
He labeled E coli DNA with a radioactive DNA precursor and at selected times the DNA was observed using autoradiography. At a certain point, a structure resembling the greek letter theta was seen. This represents the theta mode of replication.
What was the significance of Cairns Theta Mode of Replication?
This autoradiography established the fact that there are two replicating forks in DNA replication. It did not however demonstrate if replication was unidirectional or bidirectional.
What experiment and by whom established that DNA replication was bidirectional?
The experiment done by Gyurasits and Wake labeled replicating DNA for a short time with low levels of radioactive precursor followed by high levels of radioactive precursor. Both forks picked up the high radioactivity label and therefore replication is bidirectional.
Is there any organism that replicates DNA unidirectionally?
Plasmid ColE1 replicates DNA unidirectionally with only one replication fork.
What is another mechanism by which some circular DNAs can replicate?
Rolling Circle replication
WHat E Coli phage uses rolling circle replication?
Sigma-X174 and it contains a single stranded circular genome
