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14. Mutation, Repair and Recombination
Learning outcomes
When you have read Chapter 14, you should be able to
1. Distinguish between the terms ‘mutation' and ‘recombination', and define the various
terms that are used to identify different types of mutation
2. Describe, with specific examples, how mutations are caused by spontaneous errors in
replication and by chemical and physical mutagens
3. Recount, with specific examples, the effects of mutations on genomes and organisms
4. Discuss the biological significance of hypermutation and programmed mutations
5. Distinguish between the various types of DNA repair mechanism, and give detailed
descriptions of the molecular events occurring during each type of repair
6. Outline the link between DNA repair and human disease
7. Draw diagrams, with detailed annotation, illustrating the processes of homologous
recombination, gene conversion, site-specific recombination, conservative and
replicative transposition, and retrotransposition, and discuss the biological significance
of each of these mechanisms
14.1. Mutations
14.2. DNA Repair
14.3. Recombination
14.1. Mutations
Figure 14.1. Mutation, repair and recombination. (A) A mutation is a small-scale change in the nucleotide
sequence of a DNA molecule. A point mutation is shown but there are several other types of mutation, as
described in the text. (B) DNA repair corrects mutations that arise as errors in replication and as a result of
mutagenic activity. (C) Recombination events include exchange of segments of DNA molecules, as occurs during
meiosis (see Figure 5.15 ), and the movement of a segment from one position in a DNA molecule to another, for
example by transposition (Section 14.3.3).
Figure 14.2. Examples of mutations. (A)
An error in replication leads to a
mismatch in one of the daughter double
helices, in this case a T-to-C change
because one of the As in the template
DNA was miscopied. When the
mismatched molecule is itself replicated
it gives one double helix with the
correct sequence and one with a
mutated sequence. (B) A mutagen has
altered the structure of an A in the
lower strand of the parent molecule,
giving nucleotide X, which does not
base-pair with the T in the other strand
so, in effect, a mismatch has been
created. When the parent molecule is
replicated, X base-pairs with C, giving a
mutated daughter molecule. When this
daughter molecule is replicated, both
granddaughters inherit the mutation.
Figure 14.3. Mechanisms for ensuring the
accuracy of DNA replication. (A) The DNA
polymerase actively selects the correct
nucleotide to insert at each position. (B)
Those errors that occur can be corrected by
‘proofreading' if the polymerase has a 3′→5′
exonuclease activity. If the last nucleotide that
was inserted is base-paired to the template
then the polymerase activity predominates,
but if the last nucleotide is not base-paired
then the exonuclease activity is favored.
Figure 14.4. The effects of tautomerism on
base-pairing. In each of these three
examples, the two tautomeric forms of the
base have different pairing properties.
Cytosine also has amino and imino
tautomers but both pair with guanine
Figure 14.5. Replication slippage. The diagram shows replication of a five-unit CA repeat
microsatellite. Slippage has occurred during replication of the parent molecule, inserting
an additional repeat unit into the newly synthesized polynucleotide of one of the
daughter molecules. When this daughter molecule replicates it gives a granddaughter
molecule whose microsatellite array is one unit longer than that of the original parent.
Figure 14.6. 5-Bromouracil and its mutagenic effect. See the text for details
Figure 14.7. Hypoxanthine is a deaminated version of adenine. The nucleoside that
contains hypoxanthine is called inosine (see Table 10.5)
Figure 14.8. The mutagenic effect of ethidium bromide. (A) Ethidium bromide is a flat platelike molecule that is able to slot in between the base pairs of the double helix. (B) Ethidium
bromide molecules are shown intercalated into the helix: the molecules are viewed
sideways on. Note that intercalation increases the distance between adjacent base pairs.
Figure 14.9. Photoproducts induced by UV irradiation. A segment of a polynucleotide
containing two adjacent thymine bases is shown. (A) A thymine dimer contains two UVinduced covalent bonds, one linking the carbons at position 6 and the other linking the
carbons at position 5. (B) The (6-4) lesion involves formation of a covalent bond between
carbons 4 and 6 of the adjacent nucleotides.
Figure 14.10. The mutagenic effect
of heat. (A) Heat induces hydrolysis
of β-N-glycosidic bonds, resulting in
a baseless site in a polynucleotide.
(B) Schematic representation of the
effect of heat-induced hydrolysis on
a double-stranded DNA molecule.
The baseless site is unstable and
degrades, leaving a gap in one
strand.
Figure 14.11. Effects of point mutations on the coding region of a gene. Four different
effects of point mutations are shown, as described in the text. The readthrough mutation
results in the gene being extended beyond the end of the sequence shown here, the
leucine codon created by the mutation being followed by AAA = lys, TAT = tyr and ATA =
ile. See Figure 3.20 for the genetic code.
Figure 14.12. Deletion mutations. In the top sequence three nucleotides comprising a
single codon are deleted. This shortens the resulting protein product by one amino acid
but does not affect the rest of its sequence. In the lower section, a single nucleotide is
deleted. This results in a frameshift so that all the codons downstream of the deletion are
changed, including the termination codon which is now read through. See Figure 3.20 , for
the genetic code. Note that if a three-nucleotide deletion removes parts of adjacent
codons then the result is more complicated than shown here. Consider, for example,
deletion of the trinucleotide GCA from the sequence … ATGGGCAAATAT … coding for metgly-lys-tyr. The new sequence is … ATGGAATAT … , coding for met-glu-tyr. Two amino acids
have been replaced by a single, different one.
Figure 14.13. Two possible effects of deletion mutations in the region upstream of
a gene
Figure 14.14. A loss-of-function mutation is usually recessive because a
functional version of the gene is present on the second chromosome copy
Figure 14.15. A tryptophan auxotrophic mutant. Two Petri-dish cultures are shown. Both
contain minimal medium, which provides just the basic nutritional requirements for
bacterial growth (nitrogen, carbon and energy sources, plus some salts). The medium on
the left is supplemented with tryptophan but the medium on the right is not. Unmutated
bacteria, plus tryptophan auxotrophs, can grow on the plate on the left, the auxotrophs
growing because the medium supplies the tryptophan that they cannot make themselves.
Tryptophan auxotrophs cannot grow on the plate on the right, because this does not
contain tryptophan. To identify a tryptophan auxotroph, colonies are first grown on the
minimal medium + tryptophan plate and then transferred to the minimal medium plate by
replica plating (see Figure 4.18 ). After incubation, colonies appear on the minimal
medium plate in the same relative positions as on the plate containing tryptophan, except
for the tryptophan auxotrophs which do not grow. These colonies can therefore be
identified and samples of the tryptophan auxotrophic bacteria recovered from the
minimal medium + tryptophan plate.
Figure 14.16. The effect of a constitutive mutation in the lactose operator. The operator
sequence has been altered by a mutation and the lactose repressor can no longer bind to
it. The result is that the lactose operon is transcribed all the time, even when lactose is
absent from the medium. This is not the only way in which a constitutive lac mutant can
arise. For example, the mutation could be in the gene coding for the lactose repressor,
changing the tertiary structure of the repressor protein so that its DNA-binding motif is
disrupted and it can no longer recognize the operator sequence, even when the latter is
unmutated. See Figure 9.24 , for more details about the lactose repressor and its
regulatory effect on expression of the lactose operon.
Figure 14.17. Hypermutation of the V-gene segment of an intact immunoglobulin gene.
See Figure 12.15 , for a description of the events leading to assembly of an
immunoglobulin gene.
Technical Note 14.1. Mutation detection
Research Briefing 14.1. Programmed mutations?
14.2. DNA Repair
Figure 14.18. Four categories of DNA repair system. See the text for details
Figure 14.19. Repair of a nick by DNA ligase
Figure 14.20. Base excision
repair. (A) Excision of a
damaged nucleotide by a
DNA
glycosylase.
(B)
Schematic representation of
the base excision repair
pathway. Alternative versions
of the pathway are described
in the text.
Figure 14.21. Short patch nucleotide excision repair in Escherichia coli. The damaged
nucleotide is shown distorting the helix because this is thought to be one of the
recognition signals for the UvrAB trimer that initiates the short patch process. See the text
for details of the events occurring during the repair pathway.
Figure 14.22. Outline of the events involved during nucleotide excision repair in
eukaryotes. The endonucleases that remove the damaged region make cuts
specifically at the junction between single-stranded and double-stranded regions of a
DNA molecule. The DNA is therefore thought to melt either side of the damaged
nucleotide, as shown in the diagram, possibly as a result of the helicase activity of
TFIIH.
Figure 14.23. Methylation of newly synthesized DNA in Escherichia coli does not occur
immediately after replication, providing a window of opportunity for the mismatch repair
proteins to recognize the daughter strands and correct replication errors.
Figure 14.24. Long patch mismatch repair in Escherichia coli. See the text for details.
Figure 14.25. Single- and double-strand-break repair. (A) A single-strand break does not
disrupt the integrity of the double helix. The exposed single strand is coated with PARP1
proteins, which protect this intact strand from breaking and prevent it from participating
in unwanted recombination events. The break is then filled in by the enzymes involved in
the excision repair pathways. (B) A double-stranded break is more serious because the
double helix is cleaved into two segments. Various proteins bind to the broken ends,
notably Ku (see the text), to protect the ends and initiate double-strand break repair.
Figure 14.26. Non-homologous end-joining (NHEJ) in humans. (A) The repair process.
Additional proteins not shown in the diagram are also involved in NHEJ. These include the
protein kinases ATM and ATR (Section 13.3.2), whose main role may be to signal to the cell
the fact that a double-strand break has occurred and the cell cycle should be arrested until
the break is repaired. If the cell enters mitosis with a broken chromosome then part of that
chromosome will be lost, because only one of the fragments will contain a centromere,
and centromeres are essential for distribution of chromosomes to the daughter nuclei
during anaphase (see Figure 5.14 ). (B) Structure of the Ku-DNA complex. On the left is the
view looking down onto the broken end of the DNA double helix, and on the right is the
side view, with the broken end of the DNA molecule on the left. Ku is a heterodimer made
up of the Ku70 and Ku80 subunits, the numbers indicating the molecular masses in kDa.
Ku70 is colored red and Ku80 orange. The DNA molecule is shown in gray. Reprinted with
permission from Walker et al., Nature, 412, 607–614. Copyright Macmilllan Magazines
Limited.
Figure 14.27. The SOS response of Escherichia coli. See the text for details.
14.3. Recombination
Figure 14.28. The Holliday model for homologous recombination
Figure 14.29. Two schemes for initiation of homologous recombination. (A) Initiation as
described by the original model for homologous recombination. (B) The MeselsonRadding modification, which proposes a more plausible series of events for formation of
the heteroduplex.
Figure 14.30. The RecBCD pathway for
homologous recombination in Escherichia coli.
The events leading to formation of the
heteroduplex are shown. In the bottom structure
the RecA-coated DNA filament has formed a
triplex structure, which is thought to be an
intermediate in the process. See the text for
details
Figure 14.31. The role of the Ruv proteins in homologous recombination in Escherichia
coli. Branch migration is induced by a structure comprising four copies of RuvA bound to
the Holliday junction with an RuvB ring on either side. After RuvAB has detached, two
RuvC proteins bind to the junction, the orientation of their attachment determining the
direction of the cuts that resolve the structure. Reprinted with permission from Rafferty
JB et al. (1996)Science, 274, 415–421. Copyright 1996 American Association for the
Advancement of Science.
Figure 14.32. Gene conversion. One gamete contains allele A and the other contains allele
a. These fuse to produce a zygote that gives rise to four haploid spores, all contained in a
single ascus. Normally, two of the spores will have allele A and two will have allele a, but if
gene conversion occurs the ratio will be changed, possibly to 3A : 1a as shown here.
Figure 14.33. The double-strand break
model for recombination in yeast. This
model explains how gene conversion
can occur. See the text for details.
Figure 14.34. Integration of the bacteriophage λ genome into Escherichia coli
chromosomal DNA. Both λ and E.coli DNA have a copy of the att site, each one
comprising an identical central sequence called ‘O' and flanking sequences P
and P′ (for the phage att site) or B and B′ (bacterial att site). Recombination
between the O regions integrates the λ genome into the bacterial DNA.
Figure 14.35. Integrated transposable elements are flanked by short direct
repeat sequences. This particular transposon is flanked by the
tetranucleotide repeat 5′-CTGG-3′. Other transposons have different
direct repeat sequences
Figure 14.36. Replicative and conservative transposition. DNA transposons use
either the replicative or conservative pathway (some can use both). Retroelements
transpose replicatively via an RNA intermediate.
Figure 14.37. A model for the
process resulting in replicative and
conservative transposition. See the
text for details.
Figure 14.38. Transposition of
a retroelement - Part 1. This
diagram shows how an
integrated retroelement is
copied into a free doublestranded DNA version. The
first step is synthesis of an
RNA copy, which is then
converted to double-stranded
DNA by a series of events that
involves
two
template
switches, as described in the
text
Figure 14.39. Transposition of a retroelement - Part 2. Integration of the retroelement
into the genome results in a four-nucleotide direct repeat either side of the inserted
sequence. With retroviruses, this stage in the transposition pathway requires both
integrase and the DNA-PKCS protein kinase and Ku proteins that are also involved in NHEJ
(Section 14.2.4; Daniel et al., 1999).