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BIO SCI 99 Study Guide - Midterm Guide: Dna Replication, Reaction Mechanism, Leaving Group

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Lecture6. DNA Replication Part I - Study Guide / Lecture Summary
**Greyed out text is optional, but perhaps helpful to understand.
Meselson and Stahl Experiment
1. What question prompted the experiment? What was one reason some scientists thought
the semiconservative model to be an unlikely candidate?
2. Understand the design of this experiment. What was the basic concept?
3. What was the independent variable? Dependent variable?
4. How did the predicted experimental outcomes differ between the three discussed models of
DNA replication?
5. What would the experimental outcomes look like if allowed to continue through additional
cycles of replication?
6. What is an isotope?
DNA Polymerase
1. Basic reaction mechanism
a. What direction does DNA polymerase catalyze polymerization?
b. With what reaction components must the DNA polymerase be supplied in order to
carry out DNA polymerization? How does each component participate in the
c. Be able to draw out the basic mechanism of the polymerization reaction. Where and
what is the nucleophile? What is the leaving group? What bond is made and what
bond is broken? Is there any reason that the RNA polymerase mechanism would be
d. How would the reaction mechanism look if DNA synthesis were catalyzed in the
reverse direction (3->5)? What would be the nucleophile? The leaving group? What
difficulty is created by this hypothetical mode of polymerization?
e. Where do you think the energy for this reaction comes from? (I did not specifically
mention this in the lecture, but the answer can be guessed from what youve
learned so far in your biology courses)
f. How is the reverse reaction prevented?
g. How does the DNA polymerase catalyze the polymerization reaction? What roles do
each of the two magnesium ions play in the reaction?
h. Why is a different enzyme (Primase instead of DNA polymerase) used to make
primers? Why are cellular primers made with RNA?
2. Polymerase structure
a. What three domains did we discuss in class and what are their function?
b. What three active sites did we discuss in class and what are their function? (the
insertion site and postinsertion site are part of the same active site)
3. Ensuring fidelity of polymerization
a. Polymerization active site
i. How does the polymerase check that the incoming dNTP is the correct one
before catalyzing the addition of the incoming nucleotide to the 3 end of
the daughter strand?
ii. What do the terms base selection and induced fit mean with regards to
the polymerase? How do these mechanisms work in the polymerase? (think
conformational changes)
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