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CHEM-E8120 Cell Biology, Examination February 15th
Questions 1 and 2: Problem analyzing and solving, each question gives maximally 5 points
Questions 3 and 4: Essay questions, Q3 gives maximally 5 points, Q4 gives maximally 10 points
Problem solving:
First, analyze the problem. If you are unsure about certain details of the problem clearly
indicate it in your answers and mention your assumptions!
Question 1. (maximally 5 points)
The dnaB gene of E. coli encodes a helicase (DnaB) that unwinds DNA at the replication fork.
Its properties have been studied using artificial substrates like those shown in Figure 1A. In
such substrates, DnaB binds preferentially to the longest single-strand region (the largest target)
available. The experimental approach is to incubate the substrates under a variety of conditions
and then subject a sample to electrophoresis on agarose gels. The short single strand
(substrates 1 and 2) or strands (substrate 3) will move slowly if still annealed to the longer DNA
strand, but will move much faster if unwound and detached. The migration of these short single
strands can be followed selectively by making them radioactive and examining their positions in
the gel by autoradiography. The migration of the labeled single strands in the three different
substrates is shown in Figure 1B. In the absence of any treatment, the labeled strands move
slowly (lanes 4, 8, and 12); when the substrates are heated to 100°C, the labeled strands are
detached and migrate more rapidly (lanes 3, 7, and 11).
The results of several experiments are shown in Figure 1B. Substrate 1, the substrate without
tails, was not unwound by DnaB and ATP at 37°C (Figure 1B, lanes 1 and 2). When either
substrate with tails was incubated at 37°C with DnaB and ATP, a significant amount of small
fragment was released by unwinding (lanes 6 and 10). For substrate 3, only the 3’ fragment was
unwound (lane 10). All unwinding was absolutely dependent on ATP hydrolysis.
Unwinding was considerably enhanced by adding single-strand DNA-binding protein (SSB)
(compare Figure 1B, lanes 5 and 6 and lanes 9 and 10). Interestingly, SSB had to be added
about 3 minutes after DnaB; otherwise it inhibited unwinding.
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1A
1B
Figure 1. Substrates used to test the properties of dnaB (1A). Results of several experiments to measure
unwinding by dnaB. Only the single-strand fragments were radioactively labeled. Their positions are shown
by the bands in this schematic diagram. ATP was included with dnaB in all incubations, which were carried
out at 37°C (1B).
A. Shortly sketch the replication fork marking all the proteins bound to it (including SSB and
DnaB), clearly mark the orientation of the DNA (5’-, 3’-ends)
B. Why do you suppose ATP hydrolysis is required for unwinding?
C. In what direction does DnaB move along the long single-strand DNA? Is this direction more
consistent with its movement on the template for the leading strand or on the template for the
lagging strand at a replication fork?
D. Why do you suppose SSB inhibits unwinding when it is added before dnaB, but stimulates
unwinding when added after dnaB?
Question 2. (maximally 5 points)
SNAREs exist as complementary partners that carry out membrane fusions between
appropriate vesicles and their target membranes. In this way, a vesicle with a particular variety
of v—SNARE will fuse only with a membrane that carries the complementary t—SNARE. In
some instances, however, fusions of identical membranes (homotypic fusions) are known to
occur. For example, when a yeast cell forms a bud, vesicles derived from the mother cell’s
vacuole move into the bud where they fuse with one another to form a new vacuole. These
vesicles carry both v—SNAREs and t—SNAREs. Are both types of SNAREs essential for this
homotypic fusion event?
To test this point, you have developed an ingenious assay for fusion of vacuolar vesicles. You
prepare vesicles from two different mutant strains of yeast: strain B has a defective gene for
vacuolar alkaline phosphatase (Pase); strain A is defective for the protease that converts the
precursor of alkaline phosphatase (pro-Pase) into its active form (Pase) (Figure 2A). Neither
strain has active alkaline phosphatase, but when extracts of the strains are mixed, vesicle fusion
generates active alkaline phosphatase, which can be easily measured (Figure 2B).
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Now you delete the genes for the vacuolar v—SNARE, t—SNARE, or both in each of the two
yeast strains. You prepare vacuolar vesicles from each and test them for their ability to fuse, as
measured by the alkaline phosphatase assay (Figure 2B).
2A
2B
Figure 2. SNARE requirements for vesicle fusion. (A) Scheme for measuring the fusion of vacuolar
vesicles. (B) Results of fusions of vesicles with different combinations of v-SNAREs and t-SNAREs. The
SNAREs present on the vesicles of the two strains are indicated as v (v-SNARE) and t (t-SNARE).
A. What do these data say about the requirements for v-SNARES and t—SNAREs in the fusion
of vacuolar vesicles?
B. Does it matter which kind of SNARE is on which vesicle?
C. Considering the in vivo situation, what additional factors/proteins are involved in the stages
immediately preceding the fusion and docking?
Essay questions
Question 3. Essay question, maximally 5 points
Elaborate on the diverse roles of Phosphatidylinositol (PI) and its various phosphorylated forms
(PIPs) in eukaryotic cells. Discuss 2 cellular processes, where PIs are involved.
Question 4. Essay question, maximally 10 points
The unfolded proteins response is a cellular reaction to cope with the accumulation of unfolded
proteins in the Endoplasmic reticulum. Ultimately, its activation leads to the induction of the
gene expression, for example of a chaperone.
A. Give a rough sketch how the signal is relayed into the nucleus (max. 2 points).
B. Give a detailed account of the events taking place after the signal reaches the nucleus until
said chaperones reach their target destination to mitigate the stress (max. 8 points).
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