Fundamental Molecular Biology: Genomes

2016-05-10
Terry Brown
Genomes
Third Edition
Chapter 16:
Mutations and DNA Repair
Copyright © Garland Science 2007
Fundamental
Molecular Biology
Second Edition
Lisabeth A. Allison
Chapter 7
DNA Repair Pathways
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn of the
Double Helix”, oil and mixed media on canvas, © 2003
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 A mutation is an alteration in the nucleotide sequence of a DNA molecule.
Figure 16.1a Genomes 3 (© Garland Science 2007)
Mutations are of fundamental importance
• Mutations are important as the major source of

genetic variation that drives evolutionary
change.
• Mutations may have deleterious or (rarely)

advantageous consequences to an organism
or its descendants.
• Mutant organisms are important tools for

molecular biologists in characterizing the
genes involved in cellular processes.
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1) Point mutation: a single nucleotide change in a DNA molecule

Transition: Purine (A or G) to Purine
Pyrimidine (C or T) to Pyrimidine
Transversion: Purine to Pyrimidine

Pyrimidine to Purine
In humans, the ratio of transitions to transversions is approximately 2:1
- A tautomeric shift produces a transition mutation
- Transitions are less likely to result in amino acid substitutions
2) Insertion
3) Deletion
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 All cells have DNA repair enzymes to minimize mutations.
 Two types of DNA repair enzymes

1) Prereplicative enzymes: search the DNA for nucleotides with unusual
structures and replace them before replication.
2) Postreplicative enzymes: check newly synthesized DNA for errors and
correct them.
Figure 16.1b Genomes 3 (© Garland Science 2007)
16.1 Mutations
16.1.1 The cause of mutations
 Mutations arise in two ways (Mismatch mutations, mutagens).
1) Mismatch mutations: spontaneous errors in replication that evade the

proofreading function of the DNA polymerase.
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2) Mutagens: react with the parental DNA and cause a structural change.
The change affects the base-pairing capability of the altered nucleotide.
1. Errors in replication are a source of point mutations

 DNA polymerase has two mechanisms for ensuring the accuracy of DNA
replication.
1) DNA polymerase actively selects the correct nucleotide to insert at each position.
 When the nucleotide is first bound to the DNA polymerase
 When the nucleotide is shifted to the active site of the enzyme
 When the nucleotide is attached to the 3’ end of the polynucleotide
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2) DNA polymerase can proofread nucleotide selection error using 3’ to 5’

exonuclease activity.
 Sometimes, an error occurs even though DNA polymerase adds the

correct nucleotide.
 The effects of tautomerism on base pairing
 Thymine and Guanine exist as keto and enol forms.

 Adenine and Cytosine exist as amino and imino forms.
 Dynamic equilibrium is biased very much toward the keto and amino forms.
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Figure 16.4 part 1 of 3 Genomes 3 (© Garland Science 2007)
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amino-cytosine imino-cytosine
 Cytosine cannot produce mutation by tautomeric shift.
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2. Replication errors can also lead to insertion and deletion mutations
http://www.studyblue.com/notes/note/n/bio-340-exam-3-prep/deck/761340
 One or two base pairs insertion or deletion cause a frameshift mutation.

 The frameshift mutation occurs within a coding region and changes the reading
frame used for translation.
Replication slippage
 Insertion and deletion mutations are prevalent when the template DNA contains
short repeated sequences, such as microsatellities.
 Repeated sequences can induce replication slippage.
 This is the main reason why microsatellite sequences are so variable.
Figure 16.5 Genomes 3 (© Garland Science 2007)
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 Replication slippage is probably also responsible for the trinucleotide repeat

expansion disease.
 Huntington’s disease patients have more than 36 copies of CAG repeats, which
generate more polyglutamine tract and make the protein nonfunctional.
Table 16.1 Genomes 3 (© Garland Science 2007)
 During polymerization, thermal fluctuation

can dissociate DNA strands.
 The reassociation is usually perfect,
resulting in no change.
 However, occasionally, the DNA may align
unequally, due to the repeats.
 Some repeats may promote this procedure
due to stabilization of the transition state by
forming stem-loop structure from the
imperfect double helix, especially
CAG/CTG repeat, which is involved in
pathogenesis of several trinucleotide
expansion diseases.
 The longer the repeats, the higher
probability of polymerase slippage.
 It creates a positive feedback loop and
results in the increasing severity and
decreasing age of onset with each new
generation as the expansion of repeats
insertion deletion increases.
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3. Mutations are also caused by chemical and physical mutagens
1) Mutagen: a chemical or physical agent that causes

mutations
a. Base analogs
b. Agents react directly with DNA, causing structural changes
c. Agents act indirectly on DNA
2) Chemical mutagens
 Base analogues
 Deaminating agents
 Alkylating agents
 Intercalating agents
1. Base analogs
 similar enough to the standard bases of DNA to be incorporated into nucleotides.
Thymine
5 5
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 The equilibrium between the two tautomers of 5-bromouracil is shifted more

toward the rarer enol form compare with Thymine.
 5-bromouracil has more chance to base-pair with Guanine instead of Adenine.
Figure 16.6c Genomes 3 (© Garland Science 2007)
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2-aminopurine
2. Deaminating agents
 Nitrous acid: Adenine, Guanine, Cytosine

 Sodium bisulfite: Cytosine
 Adenine  Hypoxanthine (A::T  HPX::C  G::C)
 Guanine  Xanthine: blocks replication
 Cytosine  Uracil (G::C  A::U  A::T)
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3. Alkylating agents
 Ethylmethane sulfonate and
Dimethylnitrosamine add alkyl groups
to nucleotides in DNA.
 Methylation often results in modified

nucleotides and thus, point mutations.
 Alkylaiton causes crosslingking

between two strand of DNA.
 Large alkyl group prevents progress

of the replication complex.
 O6-alkylguanine causes a point

mutation or strand breakage.
 O6-alkylguanine DNA alkyltransferase

repairs O6-alkylguanine.
O6-alkylguanine DNA alkyltransferase
4. Intercalating agents
 Intercalating agents are usually associated with insertion mutations.
 The best-known mutagen is ethidium bromide.
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 Intercalating agents are flat molecules so they can slip between base pairs in the
double helix.
 This intercalation slightly unwinds the helix and hence increases the distance
between adjacent pairs.
3) Physical mutagens
 Ultraviolet radiation
 Ionizing radiation
 Heat
1. Ultraviolet radiation (260 nm)

 UV light of wavelength 260 nm is one of
physical mutagens.
 It induces dimerization of adjacent

pyrimidine bases.
 Preference is TT > CT > TC > CC.
 This dimerization causes a deletion

mutation.
 Another type of UV-induced mutation is the

(6-4) photoproduct.
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2. Ionizing radiation
 Ionizing radiation causes various mutations, such as point

mutatios, insertion, and deletion depending on the type of
radiation and its intensity.
 Some types of ionizing radiation act directly on DNA, others act

indirectly by stimulating the formation of reactive molecules such
as peroxide in the cell.
3. Heat
 Heat stimulates the water-induced cleavage of the -N-glycosidic bond,

which attaches the base to the sugar component.
 This occurs more frequently with purines than with pyrimidines and
results in an AP (apurinic/apyrimidinic) site or baseless site.
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 The baseless sugar-phosphate is unstable and rapidly degrades .
 10,000 AP sites are produced in each human cell per day.
 Cells have effective systems for repairing gaps.
 But under the certain circumstances, they are mutagenic.

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16.1.2 The effects of mutations

1. The effects of mutations on genomes
Missense mutation
Silent mutation
 Silent (Synonymous) mutations have no effect on the functioning of the genome.

 Missense (Nonsynonymouse) mutations cause amino acid changes.
 A nonsense mutation converts an amino-acid coding codon into a termination
codon.
 A readthrough mutation converts a termination codon into an amino-acid coding
codon.
frameshift mutation
 If the number of deleted or inserted nucleotides is three or a multiple of three,

then one or more amino acids are removed or added.
 If deleted amino acids are involved in active sites of enzymes or added amino
acids disrupt important secondary structure in the protein, the mutations impact
protein function.
 One or two nucleotides deletion or insertion causing frameshift mutations induces
more significant effects.
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 Sometimes, mutations on the non-coding regions affect gene expression.
1) Transcriptional regulatory sequences

 Deletion of the regulatory sequence causes uncontrolled transcription.
 Deletion of the core promoter causes no transcription
2) Splicing mutations
 Mutations on splicing donor (GT) or acceptor sequence (AG) cause that

the target intron is not removed or more frequently cryptic splicing.
 This leads to deletion of a part of the protein or frameshift mutations.
Ex) -thalassemia (hemolytic anemia): caused by mutations that lead to

cryptic splice site selection during processing of –globin transcripts.
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2. The effects of mutations on multicellular organisms
1) By inheritance
A. Somatic cell mutation
 does not pass copies of their genome to the next generation.

 is important only for the person with the mutation.
 Most somatic cell mutations have no significant effect because we have a
lot of identical cells in our body.
B. Germ cell mutation
 affects next generation.
2) By effect on function
A. Loss-of-function
 It’s the result of mutations causing reduction or abolishment of a protein activity.
 Most loss-of-function mutations are recessive.
 Single copy of functional allele can compensate for the effect of the mutation.
 Haploinsufficiency
 The organism with the haploinsufficiency mutation cannot tolerate the 50%
reduction in protein activity.
 Marfan syndrome is caused by a mutation on fibrillin gene, encoding the
connective tissue protein.
Patients with the mutation are unusually tall, with long limbs and long, thin
fingers.
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B. Gain-of-function
 Gain-of-function mutations change the gene product such that it gains a new
and abnormal function.
 The mutations are much less common and usually dominant.
 Many gain-of-function mutations are in regulatory sequences rather than in
coding regions.
 Expressed in wrong tissue.
 Overexpression of gene(s) involved in control the cell cycle.
C. Dominant negative mutations
 Dominant negative mutations have an altered gene product that acts

antagonistically to the wild-type allele.
D. Lethal mutations
 Lethal mutations are mutations that lead to the death of the organisms which
carry the mutations
3) By the time of impact
A. Delayed onset
 A mutation whose effect is not apparent until a relatively late stage in the life of
the mutant organism.
B. Nonpenetrance
 The situation whereby the effect of a mutation is never observed during the
lifetime of a mutant organism.
 Hereditary PAH caused by BMPR2 mutations has only 10~20% penetrance.
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3. The effects of mutations on microorganisms

 Bacterial mutations are usually classified by growth properties of mutant cells in
various culture media.
 Auxotrophs
 Conditional-lethal mutants
 Inhibitor-resistant mutants
 Regulatory mutants
A. Auxotrophs
 Auxotrophs can only grow on the medium with a nutrient, which is not required by
the wild type bacteria, called prototrophs.
 Tryptophan operon.
 The operon controls expression of genes involved in Trp production.
 In the presence of Trp, Trp binds to TrpR protein and inhibits transcription of Trp
operon.
 Without Trp, the TrpR protein is inactive, so that the Trp operon is expressed.
 Mutations on the genes in the system generate Trp auxotrophs.
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B. Conditional-lethal mutants
 They can survive under permissive conditions, but when they transferred to
restrictive conditions they cannot survive.
 Temperature-sensitive mutants are typical examples of conditional-lethal
mutants.
 At low temperature, temperature-sensitive mutants behave like wild-type cells,
but at high temperature the cells exhibit mutant phenotypes.
 Usually, the phenotype is caused by a mutation, which reduces the stability of a
protein.
C. Inhibitor-resistant mutants
 They are tolerant of the toxic effects of antibiotics or another type of inhibitors.
 Streptomycin resistance in E. coli results from a change in the structure of
ribosomal protein S12.
 Streptomycin binds to the S12 protein of the 30S subunit of the bacterial
ribosome and interferes the binding of formyl-methionyl-tRNA to the 30S subunit.
 It prevents initiation of protein synthesis and leads to death of bacteria.
D. Regulatory mutants
 They have mutations in promoter and other regulatory sequences.

 Constitutive mutants are included in the category.
 A mutation in the operator sequence of the lactose operon can prevent the
repressor binding.
 Lac operon is constitutively expressed all the time, even when lactose
(disaccharide sugar) is absent.
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16.1.3 Hypermutation and the possibility of programmed mutations

1. Hypermutation results from abnormal DNA repair processes
Evolution
1. Darwinian evolution: not influenced by environment, random mutation

2. Lamarckian evolution: mutations occur in response to the environment
Hypermutation
 It occurs when a cell allows high mutation rate.

 Hypermutation of the V gene segments.
 Hypermutation produces additional diversity after assembly of the intact Ig gene.
 Its mutation rate is 106~7 times higher than the background mutation rate.
Suggested mechanisms
1) Hypermutation by incorrect repair of mismatches
 Normal mismatch repair system corrects a mutation in the daughter strand.

 But at the V segments the mismatch repair system changes the nucleotide in the
parent strand.
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2) Hypermutation by conversion of cytosine to uracil
Cytosine deaminase
Uracil-DNA glycosylase
 Cytosine deaminase deaminates cytosine to produce uracil.

 Uracil-DNA glycosylase removes the Uracil and makes an AP site.
 Normal repair system fill the AP site but in hypermutation the AP sites are not
repaired.
 Any nucleotide can be inserted opposite the AP site.
2. Programmed mutations appear to support the Lamarckian theory of

evolution
 In 1943 Luria and Delbruck designed
an experiment to distinguish between
random mutation and programmed
mutation.
 They cultured E.coli in different flasks
for hours and added T1 phage.
 Only T1-phage-resistant E.coli could
survive.
 If random mutation was right, T1
resistance occurred randomly, so each
flask had different numbers of T1-
resistant colonies.
 If programmed mutation was right, T1-
resistant E.coli arose when T1 phage
was added so each culture showed
similar number of colonies.
 They found that each culture

contained a different number of T1-
resistant colonies.
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Programmed mutation
 Experiment using E.coli carrying a nonsense mutation in the LacZ

gene supported programmed mutation.
 E.coli is lactose auxotrophs, so they cannot survive on the medium having

lactose as a the only carbon source.
 To survive on the medium, E.coli should have mutation converting the

nonsense mutation codon into an amino acid codon.
 When the auxotrophs are plated on to a lactose minimal medium, the

number of colonies is significantly higher than that expected by random
mutation.
 This experiment suggests that E.coli can do programmed mutations

according to the selective pressures to which they are exposed.
Programmed mutation
 E. coli cells with a lac+1 frameshift mutation cultured on
lactose minimal medium and analyze Lac+ E. coli.
 If Lac+ E. coli occurred before exposure to the lactose
plates by random mutation they form visible colonies by
about two days.
 If Lac+ E. coli occurred after exposure to the lactose
plates by programmed mutation they form visible
colonies during the following days .
 The colonies that emerge during the following days on
the lactose medium belong to two classes.
1) Most of the early Lac+ colonies are adaptive point
mutants.
 by a recombination dependent point mutation
mechanism and produce compensatory frameshift
mutations.
2) On later days, an increasing fraction of the colonies are
not point mutants but instead carry amplification to 20–
50 direct repeats of a 7~40 kb region of DNA that
contains the lac+1 allele.
 Because of its leakiness, the lac+1 mutant genes
provides sufficient gene activity to allow growth on
lactose medium.
 Both genetic changes are adaptive not random effects.
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 The experiment cannot excludes the possibility that

the late-forming colonies arise by random mutation
and form colonies late due to their slow growing.
 The colonies formed at 2,3,4, and 5 days are

replated on the lactose medium.
 If random mutation is right, 2-days colonies form

colonies 2 days after replating and 5-days colonies
form colonies 5 days after replating.
 If programmed mutation is right, all 2,3,4,5 days

colonies form colonies 2 days after replating.
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16.2 DNA Repair
 Direct repair acts directly on damaged nucleotides and converts them to original
nucleotides.
 Excision repair involves excision of a segment of the polynucleotide containing a
damaged site and resynthesizes the region by DNA polymerase.
 Mismatch repair corrects replication errors by excision and resynthesis of the
damaged region.
 Nonhomologous end-joining is used to mend double-strand breaks.
16.2.1 Direct repair systems fill in nicks and correct some types of
nucleotide modification
1) Nick
Ionizing radiation
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2) Alkylation
 Some forms of alkylation damage are
directly reversible by enzymes that
transfer the alkyl group from the
nucleotide to their own peptide chains.
 E.coli Ada protein.

 Ada has two active domains (Ada-N
and Ada-C).
 The Ada-C directly demethylates O6-
methylguanine (O6-meG) in DNA to
regenerate guanine.
 It transfers the methyl group from O6-
meG to its active-site cysteine 321
residue.
 The resulting self-methylated Ada-C
domain is irreversibly inactivated.
 Ada-N demethylates methylphosphotriesters in DNA by transferring the methyl group
to the metalloactivated Cys38 residue.
 The self-methylated Ada becomes a gene activator.
 It binds to the promoters of the genes involved in the adaptive response to
methylating agents.
O6-methylguanine-DNA methyltransferase
(MGMT): human
 Human MGMT only removes alkyl groups from position 6 of Guanine.
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3) Photoreactivation
 UV expose generates cyclobutyl dimers and (6-4) photoproduct.
DNA photolyase
 In E.coli, DNA photolyase is activated by light with a wavelength between 300

nm and 500 nm, binds to cyclobutyl dimers, and converts them to the original
nucleotides.
 (6-4) photoproduct photolyase repairs (6-4) lesions.
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16.2.2 Excision repair
Human has only one gene involved in direct repair (MGMT)

but has at least 40 genes for excision repair
O6-methylguanine-DNA methyltransferase
(MGMT, human)
1) Single base changes

- Base excision repair
- Mismatch repair
2) Structural distortion
- Nucleotide excision repair
1. Base excision repairs many types of damaged nucleotide

 Base excision repair involves removal of a damaged nucleotide base, excision of
a short piece of the polynucleotide around the AP site, and resynthesis.
 DNA gycosylase removes a damaged base, which has relatively minor
damages.
-N-glycosidic bond
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 Each DNA glycosylase has a limited specificity.
1) Deaminated bases: uracil (cytosine), hypoxanthine (adenine)

2) Oxidation products: 5-hydroxycytosine, thymine glycol
3) Methylated bases: 3-methyladenine, 7-methylguanine, 2-methylcytosine
 Model for DNA damage recognition by 8-oxoguanine DNA glycosylase 1

(hOGG1)
1) hOGG1 first binds nonspecifically to DNA.
2) If the enzyme encounters a normal GC base pair, then:
 The G is transiently extruded into a G-specific pocket and returned to the

double helix.
3) If the enzyme encounters a oxoG-C base pair, then:
 The oxoG is extruded into the G-specific pocket and then inserted into a lesion
recognition pocket where it is excised.
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 Base excision repair pathway in mammalian cells
1) A DNA glycosylase recognizes

and excises the damaged
base.
2) An endonuclease cleaves the

phosphodiester bond either 3′
or 5′ of the abasic site.
3) An endonuclease removes a
single (short patch) or 2-10
nucleotides (long patch).
4) DNA polymerase replaces the

missing nucleotides.
5) DNA ligase seals the gap.
 Short patch base excision repair pathway
1) Glycosylase associated -lyase

makes a nick 3’ to the AP site in the
DNA
2) Apurinic/apyrimidinic endonuclease
(APE) makes a nick 5’ to the AP site
in the DNA
3) DNA pol replaces the missing

nucleotide
4) DNA ligase 3-XRCC1 complex seals

the gap.
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 long patch base excision repair pathway
1) APE makes a nick 5’ to the AP site in

the DNA
2) Then, DNA pol  or  and PCNA

displace the strand 3’ to the nick to
produce a flap of 2-10 nts in length.
3) FEN1 (flap endonuclease 1) cut at

the junction of the single to double
strand transition.
4) DNA pol  or synthesizes the

missing nucleotides
5) DNA ligase I seals the gap.
2. Mismatch repair: correcting errors of replication
 The correction of mismatched base pairs which result from DNA

polymerase errors during replication.
 A large region of DNA including the mismatch is excised.
 Mismatch repair needs to distinguish the daughter strand from the
parent strand.
 E. coli use DNA methylation to distinguish the daughter strand from
the parent strand.
1) DNA adenine methylase (Dam):adenine  6-methyadenine in

5’-GATC-3’
2) DNA cytosine methylase (Dcm): cytosine  5-methycytosine in
5’-CCAGG-3’ and 5’-CCTGG-3’
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 Parent strands are fully methylated.
 Newly synthesized daughter strands

are not methylated.
 The unmethylated state is maintained

until the mismatch repair enzymes can
recognize the daughter strands and
correct replication errors.
E. coli mismatch repair systems
1) Long patch
2) Short patch
3) Very Short patch
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 A working hypothesis of long patch

mismatch repair system (>1 kb) in E. coli.
- Methylated parental strand is black and

unmethylated daughter strand is gray lines.
1) After MutS (Green) binds to a mismatch

site, it recruits MutL (Yellow) form a MutS–
MutL–DNA ternary complex.
2) This ternary complex activates MutH (Pink)

to nick DNA 5’ to GATC site of the
unmethylated strand.
3) For UvrD (Purple) activation, MutL contacts

DNA near both the mismatch and the nicked
GATC sites, and loops out the intervening
sequence.
4) UvrD unwinds the DNA and exonuclease
removes the intervening sequence.
The EMBO Journal (2004) 23, 4134–4145 5) Completion of mismatch repair requires
DNA re-synthesis and strand ligation.
 Short patch and very short patch mismatch repair systems have similar
mechanisms using different proteins recognizing the mismatches.
 The short patch mismatch repair system excises < 10 nts in length and
begins when MutY recognizes an A-G or A-C mismatch.
 The very short patch mismatch repair system recognizes G-T

mismatches by the Vsr endonuclease.
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 Mismatch repair pathway in mammalian cells: The method of strand

discrimination in mammalian cells is currently unknown.
1) Damage recognition by the MutS/MutL complex: One model proposes that

MutS/MutL then diffuses for several thousand nucleotides either 5′ or 3′.
2) A 5′ or 3′ single-strand break is generated by EXO1 in association with clamp

PCNA and clamp loader RFC.
3) 5′→3′ or 3′→5′ progessive exonuclease activity of EXO1 removes the mismatch.
 Mismatch repair pathway in mammalian cells
4) 5′→3′ repair synthesis is mediated by DNA poly and associated

factors.
5) Ligation of the remaining gap is catalyzed by DNA ligase I.
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3. Nucleotide excision repair is used to correct more extensive types of

damage
extensive types of damage

- Intrastrand cross-links
- Large chemical attached bases
- Cyclobutyl dimers (dark repair system)
E. coli
1) Short patch nucleotide excision repair

: 12 nts
UvrB: cut the 5th phosphodiester bond

downstream
UvrC: cut the 8th phosphodiester bond
upstream
2) Long patch nucleotide excision repair

: up to 2 kb,
more extensive forms of damage
Nucleotide excision repair in mammals
1) Global genomic NER (GG-NER) corrects damage in transcriptionally

silent areas of the genome.
2) Transcription coupled NER (TC-NER) repairs lesions on the actively

transcribed strand of the DNA.
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 Nucleotide excision repair in 1) Damage is recognized by the

mammals cooperative binding of XPC, RPA, XPA,
and TFIIH.
- XPC binds first, followed by the other
proteins.
- TFIIH is a multiprotein complex including

XPB and XPD that also plays an important
role in gene transcription.
2) Unwinding of the duplex DNA is

promoted by the action of XPB and XPD
helicases.
3) The endonuclease XPG makes a 3′

incision (~6 nts), and a 5′ incision (~20
nts) is made by the endonuclease XPF-
ERCC1.
4) Repair synthesis is mediated by DNA

polymerase  or .
5) Ligation of the remaining gap in the DNA

backbone is mediated by DNA ligase I.
 Defects in nucleotide excision repair underlie human disease
 Nucleotide excision repair is the only way to repair cyclobutyl dimer and other
photoproducts.
 Mutations on the genes involved in this repair sytem cause xeroderma
pigmentosum.
 Symptoms of xeroderma pigmentosum are hypersensitivity to UV radiation,
higher mutation rate on exposure to sunlight, and skin cancer.
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 Complementation group: fibroblast cells of two different patients are fused in

vitro, and then nucleotide excision repair is determined by the uptake of
radiolabeled thymidine into DNA.
 If fibroblast cells of two different patients with the same defect, the DNA damage
is not repaired.
 If the two patients have different gene defects, the cells correct each other and
the DNA damage is repaired.
16.2.3 Repair of DNA breaks
1) Single strand break Oxidative damage
 Single strand break in DNA is repaired by PARP1 and proteins involved in

excision repair system.
 PARP1 proteins protect single-strand DNA region and prevent unwanted
recombination.
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2) Double strand break

 Double strand breaks are generated by exposure to ionizing radiation
and chemical mutagens and also occur during DNA replication.
 Most organisms have two distinct pathways for repair of double strand
breaks.
1) Homologous recombination
2) Nonhomologous end-joining (NHEJ)
Nonhomologous end-joining (NHEJ)
 Double-strand breaks are repaired through direct ligation of the

DNA ends without any requirement for sequence homology.
 In mammalian cells, double-strand breaks are primarily repaired

through NHEJ.
 This double-strand break repair process can lead to mutation.
 Two broken ends can be ligated together regardless of whether

they came from the same chromosome.
 Frequently results in insertions or deletions at the break site.
 Trade-off between repair and otherwise lethal breaks in the

genome.
41
2016-05-10
Model for mammalian Nonhomologous end-joining (NHEJ)

1) Double-strand break: induced by
ionizing radiation.
2) End recognition: broken ends are

recognized by heterodimers of
Ku70/Ku80.
3) End processing:
a. the endonuclease Artemis is
activated after it is phosphorylated
IV
by the DNA-dependent protein
kinase catalytic subunit (DNA-PKCS).
b. the activated Artemis complex then
trims excess or damaged DNA at
the break site.
c. DNA pol  and  fill-in gaps and
extend 3′ or 5′ overhangs.
4) End bridging: the ligase complex XRCC4-DNA ligase IV is recruited to the
damaged site and forms a bridge.
5) Ligation: the broken ends are ligated by the XRCC4-DNA ligase IV complex.
42
2016-05-10
16.2.4 Bypassing DNA damage during genome replication
 If the genome has suffered extensive damage, the cell will face a stark
choice between dying or attempting error-prone replication.
 E.coli chooses error-prone replication.
 Specialized low-fidelity, “error-prone” DNA polymerases transiently

replace the replicative polymerases and copy past damaged DNA.
 Typical error rates range from 10-1 to 10-3 per base pair.
 The error-prone DNA polymerases have the active site, which is more
spacious compared to the high-fidelity polymerases.
1. The SOS response is an emergency measure for coping with a

damaged genome  The SOS response is an emergency system for
coping with a damaged genome.
 When E.coli DNA polymerase III encounters

highly damaged template DNA, normal
replication process is blocked or delayed.
 It initiates the SOS response, which requires

DNA polymerase V and RecA.
 The DNA polymerase V and several copies of

the RecA protein form mutasome.
 The DNA polymesase V consists of two UmuD’

and one UmuC subunits.
 RecA proteins coat the damages region,

displace DNA polymerase III, and recruit DNA
polymerase V.
 DNA polymerase V carry out error-prone DNA

synthesis.
43
2016-05-10
 Detailed SOS response mechanism.

1) LexA repressor binds to the operators of more than 30 SOS genes including
UmuD and UmuC and keeps them repressed.
2) In the presence of SOS signal, the RecA protein forms a close-packed 'activated'
RecA filament.
3) The activated RecA* acts as a co-protease to cleave any LexA released from
low-affinity operators.
4) Further cleavage of LexA induces SOS gene expression.
2. Models for mammalian error prone replication
1) The replication machinery is arrested at a

thymine dimer: Multiple error-prone DNA
polymerases are stored in a subnuclear
compartment near the arrested replication
fork.
2) When the replication fork stalls at the

lesion, DNA pol  is replaced by one of the
error-prone polymerases.
3) When translesion synthesis has bypassed

the damage, another polymerase switch
occurs. High-fidelity DNA replication
continues.
44
2016-05-10
 DNA polymerase eta () = POLH = XPV

1) Performs translesion synthesis past TT dimers by inserting AA.
2) Has an extra wide active site that can accommodate two dNTPs instead
of one.
3) Van der Waals forces and hydrogen-bonding interactions hold the TT

dimer so that the two thymines can be paired with two adenines.
45

Fundamental Molecular Biology: Genomes

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Fundamental Molecular Biology: Genomes

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Copyright © Garland Science 2007

 A mutation is an alteration in the nucleotide sequence of a DNA molecule.

Figure 16.1a Genomes 3 (© Garland Science 2007)

Mutations are of fundamental importance

• Mutations are important as the major source of

• Mutations may have deleterious or (rarely)

• Mutant organisms are important tools for

1) Point mutation: a single nucleotide change in a DNA molecule

Transversion: Purine to Pyrimidine

Figure 16.1a Genomes 3 (© Garland Science 2007)

 All cells have DNA repair enzymes to minimize mutations.

 Two types of DNA repair enzymes

1) Mismatch mutations: spontaneous errors in replication that evade the

Figure 16.2a Genomes 3 (© Garland Science 2007)

Figure 16.2b Genomes 3 (© Garland Science 2007)

1. Errors in replication are a source of point mutations

Figure 16.3a Genomes 3 (© Garland Science 2007)

2) DNA polymerase can proofread nucleotide selection error using 3’ to 5’

Figure 16.3b Genomes 3 (© Garland Science 2007)

 Sometimes, an error occurs even though DNA polymerase adds the

 Thymine and Guanine exist as keto and enol forms.

Figure 16.4 part 1 of 3 Genomes 3 (© Garland Science 2007)

Figure 16.4 part 2 of 3 Genomes 3 (© Garland Science 2007)

Figure 16.4 part 3 of 3 Genomes 3 (© Garland Science 2007)

 Cytosine cannot produce mutation by tautomeric shift.

2. Replication errors can also lead to insertion and deletion mutations

 One or two base pairs insertion or deletion cause a frameshift mutation.

Figure 16.5 Genomes 3 (© Garland Science 2007)

 Replication slippage is probably also responsible for the trinucleotide repeat

 During polymerization, thermal fluctuation

3. Mutations are also caused by chemical and physical mutagens

1) Mutagen: a chemical or physical agent that causes

Figure 16.6a Genomes 3 (© Garland Science 2007)

 The equilibrium between the two tautomers of 5-bromouracil is shifted more

Figure 16.6b Genomes 3 (© Garland Science 2007)

Figure 16.6c Genomes 3 (© Garland Science 2007)

 Nitrous acid: Adenine, Guanine, Cytosine

Figure 16.7 Genomes 3 (© Garland Science 2007)

 Methylation often results in modified

 Alkylaiton causes crosslingking

 Large alkyl group prevents progress

 O6-alkylguanine causes a point

 O6-alkylguanine DNA alkyltransferase

 Intercalating agents are usually associated with insertion mutations.

 The best-known mutagen is ethidium bromide.

Figure 16.8a Genomes 3 (© Garland Science 2007)

Figure 16.8b Genomes 3 (© Garland Science 2007)

1. Ultraviolet radiation (260 nm)

 It induces dimerization of adjacent

 Preference is TT > CT > TC > CC.

 This dimerization causes a deletion

 Another type of UV-induced mutation is the

Figure 16.9 Genomes 3 (© Garland Science 2007)

 Ionizing radiation causes various mutations, such as point

 Some types of ionizing radiation act directly on DNA, others act

 Heat stimulates the water-induced cleavage of the -N-glycosidic bond,

Figure 16.10a Genomes 3 (© Garland Science 2007)

 The baseless sugar-phosphate is unstable and rapidly degrades .

 10,000 AP sites are produced in each human cell per day.

 Cells have effective systems for repairing gaps.

 But under the certain circumstances, they are mutagenic.

16.1.2 The effects of mutations