23 replication Flashcards
what are the 3 possible models of replication
semiconservative, conservative, dispersive
What is meselson and Stahl experiment?
- grew bacteria in 15N-containing (heavy) medium
- transfer bacteria to 14N -containing (light) medium
- remove sample at various time intervals
- seperate DNA from bacteria using equilibrium density gradiant centrifugation
What did the results look like for meselson and stahl experiment if DNA is semiconservative?
What did the results look like for meselson and stahl experiment if DNA is conservative?
What did the results look like for meselson and stahl experiment if DNA is dispersive?
which models of replication is correct?
semiconservative replication
What are the components neeed for DNA synthesis?
- a primer strand and a template strand
- incoming dNTPs (N=AGCT) are complementary to the template strand
- DNA polymerase adds deoxyribonucleotides to the free 3-OH end of the primer strand.
what direction does the newly synthesized strand grow in?
5’ to 3’
How do we know DNA synthesis is from 5’ to 3’
- Add DNA polymerase and [α-32P] reactive labelled dCTP
- digest product with spleen phosphodiestease (break 5’ ester bond) or digest for a limited time with snake venome phosphodiesterase (break 3’ ester bond)
- isolate 32P labeled product, identify base attached to radiolabel by chromotography
- found support for 5’ to 3’
what would you expect to find if snake venom phosphodiesterase (3’ end digestion) was used to treat DNA?
- after centrifuge
- check for radioactivity in the supernatant (low molecular weight product)
- if DNA is added to the 3’ end, the radioactive is released when digested with the venom
- if DNA is added to the 5’ end, radioactivity should be in the pelet and not release into supernatant
How is DNA synthesis able to proceed in 5’ to 3’ direction for both strands simultaneously?
replication fork is asymmetrical
How is the replication form assymmetrical?
- the leading strand is synthesized continuously
- synthesis of lagging strand occurs in small discontinous stretched called okazaki fragments
- each fragments needs a primer,
- After DNA synthesis, primers are removed and replaced with DNA afterwards
- DNA ligase catalyzed linkage between fragments
How were okazaki fragments found?
- label E coli with short pulse of radioactive thymidine
- extract DNA and denature DNA with alkaline to seperate DNA strands
- determine size of DNA strand by sedimentation
- found small fragments of DNA
- DNA ligase to join Okazaki fragments
How are Okazaki fragment joined and how are RNA primer joined in E coli?
- the DNA polymerase III dissociates from the DNA when it reaches the next RNA primer
- 5’ to 3’ exonuclease activity of DNA polymerase I removes the RNA primer and the 5’ to 3’ polymerase activity fills in the gap with DNA
- DNA ligase seals the DNA “nick” between adjacent fragments
What do polymerase III and polymerase I do?
III = okazaki fragment synthesis
I = removes RNA and fills in with DNA
What is the sequence of enzymes used in the joining of okazaki fragments and removal of RNA in ecoli?
DNA polymerase III, then released and DNA polymerase I is recruited, released, then DNA ligase is recruited