Download Answers chapter 9

Chapter 9 Answers
1.
The mutation rate in a species reflects a balance between the cost of having deleterious
mutations appear too frequently and the cost of having too little genetic diversity. As most
mutations are either neutral or deleterious, a high mutation rate will prove damaging to
individuals (for example, producing cancer when mutations arise in somatic tissues) and their
ability to have viable offspring (if the mutations arise in the germ line). For this reason,
evolution exerts downward pressure on the mutation rate; this pressure is reflected in the
elaborate mechanisms that exist to replicate DNA accurately and to repair any incorrectly
incorporated or damaged bases. On the other hand, reducing the mutation rate to zero would not
only require an enormous investment (to produce enough repair enzymes with sufficient activity
to detect and repair all potential mutations), but would also introduce a major evolutionary
limitation: ultimately, new mutations are the sole source of the genetic variation that natural
selection acts upon. Mutations therefore permit the appearance of new traits that can prove
successful in a particular environment or allow groups to adapt to a changing one.
While the rate at which new mutations arise partly reflects uncontrolled environmental
factors such as exposure to mutagenic compounds or radiation, the rate is also dependent on
factors that can be largely controlled. For example, the DNA repair enzymes present in the cell
could be expressed at a higher or lower level, and the repair enzymes and the proofreading
activity of DNA polymerase could be more or less accurate. Accordingly, natural selection
could act upon the mutation rate by influencing the conservation of genetic changes that affect
proofreading or DNA repair activity in any of these ways.
2.
Cells possess two major mechanisms to ensure the fidelity of DNA replication. The first
of these is provided by the proofreading activity of DNA polymerase. If a nucleotide is
incorrectly incorporated into the growing strand, a 3’to 5’ exonuclease activity present in many
polymerases can remove it, allowing the polymerase to then backtrack and insert the correct
base. Such exonucleolytic repair activity provides about a 100-fold increase in replication
accuracy.
The second mechanism is provided by the mismatch repair system. Here, mismatches are
first detected by specialized enzymes (such as MutS in E. coli), and other enzymes then cut the
newly synthesized strand on either side of the incorrectly incorporated nucleotide. The stretch of
nucleotides between the cuts (including the incorrectly incorporated one) are then removed, and
the resulting gap is filled in by DNA polymerase and DNA ligase. Different organisms use
different mechanisms to identify which strand is the newly synthesized one. For example, in E.
coli newly synthesized strands are identifiable based on the fact that they contain the
unmethylated half of the hemi-methylated GATC sites that are scattered throughout the genome.
Eukaryotes use different (and not very well understood) mechanisms to recognize the newly
synthesized strand. For example, they rely on the fact that newly synthesized lagging strands
have frequent nicks (reflecting the discontinuous synthesis of Okazaki fragments). The
mismatch repair system brings another two or three orders of magnitude to the fidelity of
replication.
3.
The rate of spontaneous mutation varies within the genome, ranging from about 10-7 to
about 10-11 per round of replication for any given site within the genome. While the source of
much of this variation remains mysterious, it is clear that certain genomic regions or types of
nucleotide sequence are especially prone to spontaneous mutation.
For example, sequences including di-, tri-, and tetra-nucleotide repeats are especially
unstable. During DNA replication, these repeats can cause slippage of the replication machinery,
leading to an alteration in the number of repeats. Sequences containing methylated cytosines are
also vulnerable to mutation, because spontaneous deamination of methylated cytosine gives rise
to a thymine. Because thymine is a natural base, the cell does not recognize it as a product of a
deamination reaction and will therefore not repair it. Adjacent thymine residues are also
relatively mutagenic, as they can form thymine dimers when exposed to ultraviolet light.
Thymine dimers are incapable of base pairing and can block the replication machinery,
potentially leading to mutation. Finally, genomic regions that lack a homologous region—such
as parts of the male Y chromosome—may be prone to increased mutation rates because they lack
any available homologous regions that could be used for recombinational repair of damaged
DNA.
4.
Examples of diseases caused by trinucleotide repeat expansion include adult (myotonic)
muscular dystrophy, Fragile-X syndrome, and Huntington’s disease.
The number of trinucleotide repeats can change during replication if the DNA polymerase
“slips,” losing its place within the repeats and restarting at the wrong position. Such slippage can
produce an increase or decrease in the number of repeats.
Expanded repeats can lead to disease in different ways, including by altering gene
expression and by introducing deleterious changes into the coding sequences of genes. In the
Fragile-X syndrome, for example, expansion of a repeat at the 5’ end of the gene affects the
promoter and leads to silencing of the gene. In a number of other cases, including Huntington’s
disease, the trinucleotide repeat expansion occurs within the coding sequence, introducing a
string of glutamate residues into the protein and rendering it toxic.
5.
In E. coli, mismatches are detected and repaired in a sequential process that involves the
enzymes MutS, MutL, and MutH. MutS first scans the DNA, looking specifically for distortions
in the DNA backbone that are characteristic of mismatched base pairs. Once MutS localizes a
mismatch, it binds to the site and recruits the MutL protein. MutL then induces the MutH
enzyme to make an incision in the newly synthesized strand (which it recognizes by virtue of it
containing the unmethylated half of hemi-methylated GATC sites). The helicase UvrD then
unwinds the DNA near the incision (and including the mismatch), and the unwound DNA is
digested by an exonuclease. The gap left by this process is filled in by DNA polymerase and
sealed with DNA ligase.
Eukaryotes use a similar system to repair mismatches, involving enzymes homologous to
MutS and MutL, although the eukaryotic system is more complex (for example, eukaryotes have
specialized MutS homologs that can specifically detect different types of replication errors). One
important difference between the systems, however, is that eukaryotes do not rely on hemimethylation of specific sites in order to recognize recently-replicated DNA. Instead, other clues
are used, such as the fact that newly synthesized lagging strands are discontinuous (because of
the synthesis of Okazaki fragments).
6.
a. Double-strand breaks (repairable by the double-strand break repair pathway)
b. Alkylation of the DNA (repairable by direct removal of the alkyl group from the base,
by base excision repair, or by nucleotide excision repair)
c. Double-strand breaks (repairable by the double-strand break repair pathway)
d. Deamination of the bases (repairable by base excision repair or by nucleotide excision
repair)
e. Alkylation of the DNA (repairable by direct removal of the alkyl group from the base,
by base excision repair, or by nucleotide excision repair)
f. Double-strand breaks (repairable by the double-strand break repair pathway)
g. Damage to bases, such as the conversion of guanine to oxoG (repairable by base
excision repair or by nucleotide excision repair)
h. Thymine dimers (repairable by photoreactivation or by nucleotide excision repair)
7.
Ethidium intercalates between the bases of DNA, causing the double helix to partially
unwind. The presence of ethidium can have serious biological consequences, because the
distortion that it makes in the DNA can lead to the introduction of small insertions or deletions
during DNA replication. The addition or deletion of even a single nucleotide can change the
reading frame of a coding sequence and thereby destroy the function of the encoded protein.
8.
Alkylating agents such as EMS tend to produce point mutations into the DNA, because
alkylated bases are subject to mispairing events, which can lead to permanent nucleotide changes
if the damaged bases are not repaired prior to DNA replication. One prominent example of base
alkylation leading to mutation is the methylation of guanine at the oxygen at carbon atom 6,
producing O6-methylguanine. O6-methylguanine can pair with thymine (instead of cytosine),
changing a G:C base pair into an A:T base pair if the error is not detected in time.
Unlike EMS, X-rays produce double-stranded breaks in the DNA. Accordingly, X-rays
are generally used to induce chromosomal rearrangements such as deletions, inversions, and
translocations.
9.
Because damaged bases are buried within the double helix, they cannot be easily accessed
for repair by the DNA glycosylase enzymes of the base excision repair system. Glycosylases get
around this problem by flipping the bases out so that they project away from the double helix and
into the active site of the enzyme. Apparently, the DNA can withstand this flipping of the bases
without suffering excessive distortion, which might otherwise make the mechanism prohibitively
costly in terms of energy.
10.
The first step in base excision repair involves the recognition of the damaged base by one
of the numerous glycosylase enzymes present in the cell. Upon spotting the damaged base, the
glycosylase (which is specific for the particular type of damage) removes the base by cleaving
the glycosidic bond connecting it to the sugar component of the nucleotide. This leaves an
abasic sugar, which is subsequently removed by endonuclease enzymes. Finally, the gap left in
the DNA by the endonucleolytic cleavage is repaired by DNA polymerase (using the undamaged
strand as a template) and DNA ligase.
If the excision repair system fails to detect a damaged base prior to replication, a backup
mechanism exists that can intervene before a point mutation is permanently established in the
genome. Specifically, the cell contains certain glycolases that can recognize the mismatches that
result from the replication of damaged bases and selectively remove the nucleotide opposite the
damaged base. For example, one such glycosylase can recognize base pairs between oxoG and
A and specifically remove the incorrectly incorporated (but undamaged) A. Another glycosylase
targets T:G base pairs, a potential consequence of the spontaneous deamination of methylated
cytosines.
11.
Nucleotide excision repair in E. coli is carried out by four proteins. Three of these, UvrA,
UvrB, and UvrC, work together as a complex to scan the DNA for damaged nucleotides and to
make incisions in the DNA surrounding any identified lesions. Within this complex, UvrA
detects distortions in the DNA that are indicative of lesions, UvrB melts the DNA surrounding
any identified lesions, and UvrC cleaves the lesion-containing strand on either side of the defect.
The fourth protein, a helicase called UvrD, then unwinds the cleaved DNA to release the short
lesion-containing fragment. This leaves a gap in the DNA that is then filled in by DNA
polymerase and DNA ligase. Eukaryotes use a similar system, although it is more complex and
involves a greater number of proteins.
Individuals with xeroderma pigmentosum are especially sensitive to sunlight, and are
particularly susceptible to developing skin lesions such as cancer. This disease can be caused by
mutations in any number of genes involved in the nucleotide excision repair pathway, including
genes involved in the detection of lesions, in the melting of the DNA at the site of any detected
lesions, and in the cleavage of DNA at positions flanking the lesion.
12.
DNA polymerases that are blocked by unrepaired lesions in the DNA have a couple of
options for bypassing the damage and continuing replication. One possibility is use
recombinational repair, which allows the polymerase to replicate past the lesion using the other
daughter molecule of the replication fork as a template. A second option involves translesion
synthesis, a process that uses specialized polymerases to synthesize DNA in a templatedependent but base pair-independent manner. Translesion synthesis allows the replication fork
to bypass the lesion and to continue replicating using the lesion-containing strand as a template.
One disadvantage of this system, however, is that the nucleotides incorporated by the specialized
polymerases are not selected using Watson-Crick base pairing rules, making the process highly
error-prone.
Translesion synthesis is the option of last resort, because its reliance on polymerases that
carry out base pair-independent synthesis means that it involves a high mutation rate. Still, it is
better than nothing because a failure to complete replication (a likely consequence if the
replication fork were stalled indefinitely) would introduce even greater problems when the cell
attempts to pass through mitosis.