Discussion Problems – Fall 2023 - Week 4 (Polymerases)
Group1
Rev1 is a specialized DNA polymerase
which can only incorporate a C opposite to a
G ( it does not incorporate G, A or T in front
of a G, and does not incorporate any
nucleotides opposite to an A, T or C). The
crystal structure of Rev1 in complex with a
primer-template duplex and with dCTP has
been obtained.
The template contains a G at its 5’end, and
the primer is only one nucleotide smaller
than the template. A view of the active site is shown on the top. P
atoms are colored in purple.A and B are two divalent cations A
more detailed view of the molecular environment surrounding the
“incoming dCTP” is also shown on the right.
A- What prevents the substrates from being used by the enzyme?
B- What aspect of the active site of the Rev1 DNA polymerase is similar to other DNA
polymerases?
C- Assuming Rev1 uses a steric gate mechanism, where would the amino acid involved as a
steric gate be located?
D-Using the two pictures of the active site, explain the specificity of Rev1 for dCTP and compare
the base selection mechanism to the mechanisms used by the DNA polymerase studied in class.
Group2
The polymerization activity of a DNA polymerase (wild-type version =
Parental enzyme) or a version carrying a Tyrosine to Alanine
mutation at position 416 (Y416A) is measured in the presence of a
DNA template, a primer and all 4 dNTPs (y-axis, x-origin). Numbers
represent arbitrary polymerization activity. In separate experiments,
the enzymes are also incubated with the same components, but also
increasing concentrations of either dCDP or rCDP nucleotides and
the dNTP polymerization rates measured and plotted on the y-axis as
a function of the concentration of dCDP or rCDP.
A – Describe and explain the impact of increasing dCDP or rCDP on
the activities of the parental enzyme.
(make sure you differentiate description and explanation)
B – Describe and explain the impact of increasing dCDP or rCDP on the activities of the Y416A
enzyme.
C – based on these results propose a function for residue 416 in the mechanism of the enzyme –
your answer should fit into 1-2 sentences
Group3
A 22 nucleotide long DNA template is annealed
21nt primer
to a radiolabeled 21 nucleotide primer. The 5- 5’* 3’
terminal nucleotide of the template can be a G or 3’ X5’ (X=A,G,C, T)
22nt template
A:
These template-primer duplexes are incubated with either DNA Polymerase I, or with
a eukaryotic DNA Polymerase from the Y family of polymerases (h). The enzymes are
provided with no nucleotide (-), or with either or these 5 nucleotides: dGTP (G), dATP
(A), dTTP (T), dCTP (T), or difluorotoluene nucleoside triphosphate (F).
DNA Polymerase I
DNA Polymerase h
A- What is the purpose of using F in this experiment ?
B – Used the results of this experiment to explain the similarities and differences regarding the
fidelity mechanisms of DNA Polymerase I and DNA Polymerase h
Group 4.
In this experiment, researchers analyze the
extension of a primer annealed to a 200 nucleotide
long template by different DNA polymerases. The
first position in the template after the primer is an A.
For each DNA polymerase, they incubate the
polymerase and the template-primer duplex with
either dTTP alone, or with all dNTPs for a short
time (1 minute). The migration of the unextended
primer is also shown for most polymerases. Only
the primer is visible and the intensity of the signal is
proportional to the amount of product obtained.
1- Describe the pattern of extension generated by
each of these DNA polymerases in the presence of
all dNTPs only and compare them to each other.
What information does this experiment provides
regarding the properties of each of these
polymerases?
2- Describe the pattern of extension generated by each of these DNA polymerases in the
presence of only dTTP and compare them to each other. Why do you think it was important to
perform this experiment considering the results obtained previously?
3 – Assume Dbh is a bacterial DNA polymerase. Propose two modifications in the conditions in
which the experiment performed with Dbh is done, which would result in a pattern more similar to
the one observed for Taq or Pfrex.
