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
What is the joining of okazaki fragments and removal of RNA primer like in eukaryotes?
- DNA polymerase displaces the RNA primer and DNA from the template strand
- flap endonuclease (fen1) cleaves the displaced DNA strand
- DNA ligase seals the DNA “nick” between adjacent fragments
What does high fidelity of DNA replication mean?
polymerase recognizes the correct nucleotide: mismatched base pair do not fit the active site in the polymerase well
What is misincorporated base removed by in DNA replication?
removed by 3’ to 5’ exonuclease (proofreading) activity of DNA polymerase
polymerase repositions the mispaired 3’ terminus into the 3’ to 5’ exonuclease site and use that site to hydrolyze the mispairing with exonuclease
what is used to hydrolyze mispaired bases?
exonucleases
What is the origin of replication?
site of intiation of replication
How is replication fork movement bidirectional?
DNA replicates in both directions, forming 2 replication forks from each origin of replication
at each replication fork, DNA synthesis is continuous on one strand (leading strand) and discontinuous (lagging strand) on the other strand
why is the replication form movement bilateral?
to accelerate the process
Are there multiple origins of replication on a single eukaryotic chromosome?
Yes
* New DNA formed
* then they label cells with a pulse of [3H]dTTP, first with low then high specific activity
* found denser label indicates areas more recently replicated - the replication forks
what is the replisome?
proteins involved in DNA replication
what are the 3 phases of DNA replication?
- initiation
- elongation
- termination
what happens at initiation of DNA replication?
- initator protein binds to the origin of replication, opens up the double helix and recruits helicases
- DNA helicases unwind the helix to expose single-stranded DNA, which is coated with ssDNA binding proteins
- Primase adds RNA primers to provide 3’-OH ends for DNA synthesis
what happens at elongation of DNA replication?
- DNA polymerase is loaded onto DNA via the clamp loader and the sliding clamp
- the replication machinery moves along the DNA, adding nucleotides in a 5’ to 3’ direction through complementary base pairing
what happens at termination phase of DNA replication?
- termination occurs when 2 different forks meet or when the fork reaches the end of a lienar chromosome
- replication complexes are dissambled
- RNA primers are removed and replaced with DNA
- DNA ligase connects adjacent strands
what does the movement of growing replication form during DNA replication induce? How are they removed?
the formation of supercoilingin duplex DNA ahead of the fork
the supercoils are removed by DNA topoisomerases
what is the role of initiator protein?
binds to origin of replication
what is the role of helicase?
unwinds double-stranded DNA
what is the role of single strand binding protein?
keeps template DNA in single strand
what is the role of primase?
synthesis of primers
what is the role of topoisomerase?
relieves torsional strain
what is the role of DNA polymerases?
one for DNA replication; others for primer removal and DNA repair
what is the role of sliding clamp?
help DNA polymerase to stay on the template
what is the role of ligase?
join okazaki fragments
what is the end replication problem in linear chromosomes?
- chromosomes get shorten progressively in each generation
- because the 5’ single strand gap cannot be filled after removal of RNA primer
what is a solution to the end replication problem in linear chromosomes?
telomeres - to extend the ends of linear chromosome
* add repeating 6-mers e.g. TTGGGG at end of chromosome
* telomerase recognize this as a signal and will come and extend the 3’ single-stranded DNA end, using internal RNA molecule as template
* extended 3’ single-stranded DNA is filled in by RNA priming and DNA synthesis
* after the process is sticky end, and there are nucleases in cell that can easily digest the single strand DNA, so telomere-binding proteins to protect the ends
continuous process of adding and shortening of DNA from RNA removal. telomerase come in to work once and add a few copies for each round of replication
