Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

CHAPTER 5 END-OF-CHAPTER PROBLEMS
297
(A)
(B)
50 µm
Figure Q5–2 Autoradiographic investigation of DNA replication in cultured
cells (Problem 5–11). (A) Addition of 3 H-labeled thymidine immediately
after release from the synchronizing block. (B) Addition of 3 H-labeled
thymidine 30 minutes after release from the synchronizing block.
Problems p5.15/5.11/Q5.2
bubbles, the daughter duplexes lie side by side and cannot
be distinguished from each other.
You pretreat the cells to synchronize them at the
beginning of S phase. In the first experiment, you release
the synchronizing block and add 3 H-thymidine immediately.
After 30 minutes, you wash the cells and change the
medium so that the total concentration of thymidine is the
same as it was, but only one-third of it is radioactive. After
an additional 15 minutes, you prepare DNA for autoradiography.
The results of this experiment are shown in Figure
Q5–2A. In the second experiment, you release the synchronizing
block and then wait 30 minutes before adding
3 H-thymidine. After 30 minutes in the presence of 3 H-thymidine,
you once again change the medium to reduce the
concentration of radioactive thymidine and incubate the
cells for an additional 15 minutes. The results of the second
experiment are shown in Figure Q5–2B.
A. Explain why, in both experiments, some regions of
the tracks are dense with silver grains (dark), whereas others
are less dense (light).
B. In the first experiment, each track has a central
dark section with light sections at each end. In the second
experiment, the dark section of each track has a light section
at only one end. Explain the reason for this difference.
C. Estimate the rate of fork movement (μm/min) in
these experiments. Do the estimates from the two experiments
agree? Can you use this information to gauge how
long it would take to replicate the entire genome?
5–12 If you compare the frequency of the sixteen possible
dinucleotide sequences in the E. coli and human
genomes, there are no striking differences except for one
dinucleotide, 5ʹ-CG-3ʹ. The frequency of CG dinucleotides
in the human genome is significantly lower than in E. coli
and significantly lower than expected by chance. Why do
you suppose that CG dinucleotides are underrepresented
in the human genome?
5–13 With age, somatic cells are thought to accumulate
genomic “scars” as a result of the inaccurate repair of double-strand
breaks by nonhomologous end joining (NHEJ).
Estimates based on the frequency of breaks in primary
human fibroblasts suggest that by age 70, each human
somatic cell may carry some 2000 NHEJ-induced mutations
due to inaccurate repair. If these mutations were
distributed randomly around the genome, how many protein-coding
genes would you expect to be affected? Would
you expect cell function to be compromised? Why or why
not? (Assume that 2% of the genome—1.5% protein-coding
and 0.5% regulatory—is crucial information.)
5–14 Draw the structure of the double Holliday junction
that would result from strand invasion by both ends of the
broken duplex into the intact homologous duplex shown
in Figure Q5–3. Label the left end of each strand in the Holliday
junction 5ʹ or 3ʹ so that the relationship to the parental
and recombinant duplexes is clear. Indicate how DNA
synthesis would be used to fill in any single-strand gaps in
your double Holliday junction.
5′ 3′
5′ 3′
Figure Q5–3 A broken duplex with
single-strand tails ready to invade
an intact homologous duplex
(Problem 5–14).
5–15 In addition to correcting DNA mismatches, the
mismatch repair system functions to prevent homologous
recombination Problems p5.39/5.27/Q5.2/Q5.3
from taking place between similar but not
identical sequences. Why would recombination between
similar, but nonidentical sequences pose a problem for
human cells?
5–16 Cre recombinase is a site-specific enzyme that
catalyzes recombination between two LoxP DNA sites.
Cre recombinase pairs two LoxP sites in the same orientation,
breaks both duplexes at the same point in each LoxP
site, and joins the ends with new partners so that each
LoxP site is regenerated, as shown schematically in Figure
Q5–4A. Based on this mechanism, predict the arrangement
of sequences that will be generated by Cre-mediated
site-specific recombination for each of the two DNAs
shown in Figure Q5–4B.
(A)
BREAK
REJOIN
(B)
a b c d a b c d
Figure Q5–4 Cre recombinase-mediated site-specific recombination
(Problem 5–16). (A) Schematic representation of Cre/LoxP site-specific
recombination. The LoxP sequences in the DNA are represented by
triangles that are colored so that the site-specific recombination event
can be followed more readily. In reality their DNA sequences
are identical. (B) DNA substrates containing two arrangements of
LoxP sites.
Problems p5.43/5.32/Q5.4