the ____ ______ are the templates for DNA replication
parental strands
What are the three potential mechanisms for DNA replication?
Conservative
Semiconservative
Dispersive
Conservative
Semiconservative
Dispersive
The Melselson-Stahl Experiment (1958)
1.culture E. coli in [15N]NH4Cl-containing (heavy) medium
2.transfer bacteria to growth [14N]NH4Cl-containing (light) medium
3.remove samples at different times and extract DNA
4.subject DNA to cesium chloride density gradient centrifugation
What did the Melselson-Stahl Experiment prove?
proved semiconservative replication-parental strands separate and distribute to daughter strands
Diagram explaining the Melselson-Stahl Experiment outcome
centrifugation
heavier at the bottom, lighter at the top
What would the 3rd generation results look like in nativegradient? Bands:
C.
Discovery of DNA polymerase:
who discovered it?
Arthur Kornberg (1955)
1. Isolation
a.) dNTP's as precursors
i.end products of biosynthesis
ii.ATP -> AMP + Ppi
b)Polymerase activity assay:
i.DNA ppts in acid; dNTP's do not
c)E. coli as expt'l. organism (rapid growth)
d)Fractionated cell extracts
2. Catalyzes Polymerase Reaction
3. Mechanistic features
a)requires template strand
b)requires primer strand w/ free 3’-OH
c)ALWAYS 5’ to 3’ chain growth
d)PPi hydrolysis to drive rxn to completion
4. Polymerase catalytic mechanism
a)Nucleophilic attack by 3’-OH on a-phosphate
b)Two Mg++ mechanismi.1st to activate 3’-OHii.2nd to stabilize leaving group
c)Three conserved Asp
5. Enzymatic activities:Klenow domain:
a)Polymerase (5’ to 3’)
b)Exonuclease (3’ to 5’)
a)Exonuclease (5’ to 3’)
Domain 1
What should we consider when thinking about how Pol I manages 5’-3’ polymerase vs. 3’-5’ exonuclease(proofreading) activity?
1. Consider structural mechanism of polymerization reaction
2. Consider structural affect of incorrect bp’ing
DNA Polymerisation
(dNMP)n-1 + dNTP → (dNMP)n + PPi
_________ is the reverse reaction of DNA polymerisation
Pyrophosphorolysis
(dNMP)n + PPi →(dNMP)n-1 + dNTP
However, _____ ≠ _________
proofreading
pyrophosphorolysis
DNA polymerases can have both 5’ to 3’ and 3’ to 5’ exonuclease activity
•What does 5’ to 3’ and 3’ to 5’ mean?
•For each question below, does it refer to:
(A) 5’ to 3’ activity
(B) 3’ to 5’ activity
(C) Both
1. Generally used during DNA replication
2. Results in phosphodiester bond breakage
5' to 3' and 3' to 5' is the direction
1. A
2. B
?
Polymerization: _’-_’ DNA polymerase activity
5’-3’
Your reference strand is the strand which is affected by the DNA polymerase activity!
Proofreading: _’-_’ DNA exonuclease activity
3’-5’
Your reference strand is the strand which is affected by the DNA polymerase activitywhich is affected by the DNA polymerase activity!
Removal of RNA primer: _'-_’ exonuclease activity
5'-3'
Your reference strand is the strand which is affected by the DNA polymerase activity!