DNA damage, repair, and recombination

Mutagenesis
DNA damage
DNA repair
Recombination
 Mutagenesis

Mutation

Replication Mutagenesis
Fidelity

Mutagens

back
Mutation: Mutations are (heritable) permanent
changes in the base sequence of DNA. Point
mutations may be transitions (e.g. G:C—A:T) or
transversions (G:C—T:A). Deletions and insertions
involve the loss or addition of NT and cause
frameshifts in genetic code reading.
Silent mutations have no phenotypic effect, while
missense and nonsense mutations change the
amino acid sequence of the encoded protein.
 Mutation

Reasons
• Spontaneous errors in DNA replication or meiotic
recombination
• A consequence of the damaging effects of physical or
chemical mutagens on DNA

Various mutations and the phenotypic effects:

Point mutation: transition, transversion
Insertions or deletions: Silent mutation
Missense mutation
frameshift mutations Nonsense mutation

back
 Point mutation: a single base change

Transition : Purine or pyrimidine is replaced by

the other
AG T C

Transversion: a purine is replaced by a

pyrimidine or vice versa
A T or C T  A or G
G T or C C  A or G

back
•Noncoding DNA
•Nonregulatory DNA Silent mutation OK (no effect)
•3rd position of a codon

Missense mutation
OK (no effect)
Coding DNA → altered AA
Bad (lethal)

Coding DNA → stop codon

→ truncated protein Nonsense mutation Bad (lethal)

back
 Insertions or deletions
The addition or loss of one or more bases in a
DNA region
Causes

Frameshift mutations
The ORF of a protein encoded gene is changed
so that the C-terminal side of the mutation is
completely changed.

Bad (lethal)
back
 Replication fidelity
Important for preserve the genetic information
from one generation to the next

Spontaneous mutation rate

very low; one error per 1010 base (E. coli)

Molecular mechanisms to keep the replication fidelity

1. DNA polymerase: Watson-Crick base pairing
2. DNA polymerase: 3’→ 5’ proofreading
3. Only DNA Replication in the 5'-to-3‘ Direction Allows
efficient Error Correction
4. RNA priming: proofreading the 5’ end of the lagging strand
5. Mismatch repair
back
• to be consistent with a 2 nm width, a
purine on one strand must pair (by H-
bonding) with a pyrimidine on the other
strand
• base structure dictates which pairs of
bases can form hydrogen bonds
Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width

consistent with X-ray data
 fewer than one in 1000 accidental base changes in
DNA results in a permanent mutation; the rest are
eliminated with remarkable efficiency by DNA
repair.
 Proofreading
by
E. coli polymerase

back
1. Deamination: (C → U and A→ hypoxanthine)
2. Depurination: purine base (A or G) lost
3. T-T and T-C dimers: bases become cross-
linked, T-T more prominent, caused by UV
light (UV-C (<280 nm) and UV-B (280-320 nm)
4. Alkylation: an alkyl group (e.g., CH3) gets added
to bases; chemical induced; some harmless,
some cause mutations by mispairing during
replication or stop polymerase altogether
5. Oxidative damage: guanine oxidizes to 8-oxo-guanine,
also cause SS and DS breaks
6. Replication errors: wrong nucleotide (or modified nt)
inserted
7. Double-strand breaks (DSB): induced by ionizing
radiation, transposons, topoisomerases, homing
endonucleases, and mechanical stress on
chromosomes
Physical mutagens: Ionizing (e.g. X– and r-
rays) and nonionizing (e.g. UV) radiation produce a
variety of DNA lesions. Pyrimidine dimers are the
commonest product of UV irradiation.
Chemical mutagens: Base analogs can
mispair during DNA replication to cause mutations.
Nitrous acid deaminates cytosine and adenine.
Alkylating and arylating agents generate a variety of
adducts that can block transcription and replication
and cause mutations by direct or, more commonly,
indirect mutagenesis. Most chemical mutagens are
carcinogenic.
 Mutagens
Physical mutagens
High-energy ionizing radiation: X-rays and g-rays →
strand breaks and base/sugar destruction
Nonionizing radiation : UV light→ pyrimidine dimers

Chemical mutagens (Most of them are carcinogenic)

Base analogs: direct mutagenesis
Nitrous acid: deaminates C/A to produce U
Alkylating agents
Lesions-indirect mutagenesis
Arylating agents

back
Base analogs: derivatives of the normal bases
incorporated in DNA, altering base pairing properties.

Nitrous acid: deaminates C to produce U, resulting in →

A·U , G·C

back
 Mutagenesis

Direct mutagenesis: If a base analog or

modified base whose base pairing
properties are different from the parent
base is not removed by a DNA repair
mechanism before passage of a replication
fork, then an incorrect base will be
incorporated. A second round of
replication fixes the mutation permanently
in the DNA
 OH AGCTTCCTA
Br TCGAAGGAT
H
:G 1. Base analog
O incorporation
AGCTBCCTA
enol form TCGAAGGAT
2. 1st round
of replication
AGCTTCCTA AGCTBCCTA
Br TCGAAGGAT TCGAGGGAT
H
:A 3. 2nd round
O of replication
AGCTBCCTA AGCTCCCTA
Keto form
TCGAAGGAT TCGAGGGAT
5-BrU
A·T→G·C transition
back
Indirect mutagenesis: Most lesions in
DNA are repaired by error-free directed
reversal or excision repair mechanisms
before passage of a replication fork. If this
is not possible, trans-lesion DNA synthesis
may take place and one or more incorrect
bases become incorporated opposite the
lesion. Proofreading may be suppressed
during this process.
 DNA Damage

Oxidative damage DNA lesions

Bulky adducts

Normal condition UV light

Increased by (physical mutagens)
ionizing radiation Alkylation Carcinogen
(physical mutagens) (Chemical mutagens)

Alkylating agents
(Chemical mutagens)

back
DNA lesions: The chemical reactivity of
DNA with exogenous chemicals or
radiation can give rise to changes in its
chemical or physical structure. These may
block replication or transcription and so
be lethal, or they may generate mutations
through direct or indirect mutagenesis.
The chemical instability of DNA can
generate spontaneous lesions such as
deamination and depurination.
 DNA lesions
An alteration to the normal chemical or physical
structure of the DNA

Physical distortion of
the local DNA structure
Allowed to Remained in the DNA
Blocks replication
And/or transcription Living cell
A mutation could become fixed
Lethal by direct or indirect mutagenesis
(cell death)
Mutagenic

back
 DNA lesions
Chemical features of bases and DNA lesion

After OH group attack, G change into

8-oxoguanine, 2-oxoadenine back
 Spontaneous DNA lesions

1. Inherent chemical reactivity of the DNA

2. The presence of normal, reactive chemical
species within the cell

1. Deamination :
• C→U
• methylcytosine →T, hard to detect
2. Depurination : break of the glycosylic bond, non-
coding lesion=Lose genetic information.
3. Depyrimidine : less frequent than
depurination
back
 Cytosine deamination and repair

deamination

--ATGCTACG-- --ATGUTACG--
--TACGATGC-- --TACGATGC--
Uuracil DNA glycosylate by glycosylase
U
--ATGCTACG-- --ATG TACG--
--TACGATGC-- --TACGATGC--
back
 Oxidative damage
The presence of reactive oxygen species (ROS) in all aerobic cells.
Superoxide hydrogen peroxide
Hydroxyl radical (•OH)

Oxidation products
The levels of oxidation can be increased by hydroxyl radicals
from the radiolysis of H2O caused by ionizing radioation.
back
 Alkylation
Electrophilic chemicals which readily add alkyl groups to
various positions on nucleic acids

MMS ENU

back
 Alkylation
Electrophilic alkylating agents such as
methylmethane sulfonate (MMS) and
ethylnitrosourea (ENU) can modify nucleotides
in a variety of positions.

Most lesions are indirectly mutagenic, but O6-

alkylguanine is a directly mutagenic lesion as it
can base-pair with thymine during replication.

Some of these lesions are potentially lethal as

they can interfere with the unwinding of DNA
during replication and transcription.
 Bulky adducts

Bulky lesions such as pyrimidine

dimers and arylating agent adducts
distort the double helix and cause
localized denaturation.

This disrupts the normal functioning

of the DNA.
 Bulky adducts
Cyclobutane pyrimidine dimer
Local denaturation of DNA

Bulky lesion

Disrupt replication and

transcription

Guanine adduct of benzo[a]pyrene

Aromatic arylating
agents

Covalent adducts
back
DNA repair

Photoreactivation

Alkyltransferase

Exision repair

Mismatch repair

Hereditary (repair defects)

back
 Photoreactivation

Monomerization of cyclobutane pyrimidine

dimers by DNA photolyases in the presence of
visible light

Direct reversal of a lesion and is error-free

back
 Alkyltransferase
Removes the alkyl group from the directly mutagenic O6-
alkylguanine which can base-pair with T. the alkyl group is
transferred to the protein itself and inactivate it.
Direct reversal of a lesion and is error-free

Adaptive response to alkylating agents

The response is induced by the alkylating agents

back
 Excision repair

Repair a wide variety of lesion and is essentially error-free

Nucleotide excision repair

Base excision repair

back
 Excision repair

In nucleotide excision repair, an endonuclease makes

nicks on either side of the lesion, which is then removed to
leave a gap. This gap is filled by a DNA polymerase, and
DNA ligase makes the final phosphodiester bond.

In base excision repair, the lesion is removed by a

specific DNA glycosylase. The resulting AP site (abasic
site) is cleaved by an AP endonuclease. The resulting
single-strand break can then be processed like nucleotide
excision repair.
Nucleotide excision repair

back
 Nucleotide
excision repair

E. coli excinuclease is called the ABC excinuclease: composed of UvrA, UvrB, and
UvrC. DNA helicase is encoded by UvrD
Base excision repair

DNA glycosylases cleaves N-glycosylic bond

AP endonuclease cleaves apurinic or

apyrimidine site

DNA polymerase 3’→5’ exonuclease activity

& polymerase activity

DNA ligase

back
 Mismatch repair

Replication errors which escape proofreading

have a mismatch in the daughter strand.
Hemi-methylation of the DNA after replication
allows the daughter strand to be distinguished
from the parental strand. The mismatched
base is removed from the daughter strand by
an excision repair mechanism
 Mismatch repair

methylation of the A in GATC

of the parental strand but not of the
daughter strand right after replication

MutL/MutS recognize the mismatched

base pair and the nearby GATC

DNA helicase II, SSB, exonuclease I

remove the DNA fragment including
the mismatch

DNA polymerase III & DNA

ligase fill in the gap
back
1. Mismatch repair
Mismatch repair (cont). Methyl directed mismatch repair:
nick on non-methylated strand
 When DNA Damage Is Extensive
Repair Becomes Error-Prone
Higher levels of DNA damage, however, effectively inhibit DNA
replication and trigger a stress response in the cell involving a
regulated increase (induction) in the levels of a number of proteins.
This is called the SOS response. Some of the proteins induced,
such as the UvrA and UvrB proteins, have roles in normal DNA
repair pathways. A number of the induced proteins, however, are part
of a specialized replication system that can replicate past the DNA
lesions that block DNA polymerase III. Because proper base pairing
is often impossible at the site of a lesion, this tran-slesion
replication is error-prone. The resulting increase in mutagenesis
does not contradict the general principle that replication accuracy is
important (the resulting mutations actually kill many cells). This is
the biological price that is paid, however, to overcome the general
barrier to replication and permit at least a few mutant cells to survive.
back
Hereditary repair defects:
Xeroderma pigmentosum (XP) is an
autosomal recessive disorder characterized
phenotypically by extreme sensitivity to
sunlight and a high incidence of skin tumors.
XP sufferers are defective in the NER of bulky
DNA damage, including that caused by UV
light. Defects in at least nine different genes
can cause XP, indicating the complexity of
excision repair in mammalian cells.
Xeroderma pigmatosum
• Rare, recessive disease (fewer than 1,000 cases known worldwide)
Wide range of symptoms
blind ness and deafness
blistering or freckling on minimu m sun expos ure
developmental disabili ties
dwarfism and hypergonadism
increased skin and eye cancers
mental retardation
• Defect in nucleotide excision repair.

• Patients are very sensitive to UV rays - high rate skin cancers.

DNA damage is cumulative and irreversible

• Cells cultured from patients are unable to carry out nucleotide

excision repair; if you shine UV lights on these cells, a thymine dimers
form and are unable to be removed.

• Mutations in nine different genes can lead to this disease (XP genes).
Some have been cloned:
XP gene E. coli homolog function
XPA uvrA,
XPF uvrB Endon uclease (5’ of lesion)
XPG uvrC. Endon uclease (3’ of lesion)
XPD uvrD helicase
XPB uvrD helicase
XP and cancer incidence
Recombination

Homologous recombination
Diploid eukaryotes: crossing over
Haploid prokaryotes: Holliday model
DNA repair in replication fork
Site-specific recombination

Transposition

back
Homologous recombination:
The exchange of homologous regions
between two DNA molecules occurs
extensively in eukaryotes during meiosis.

In prokaryotes, recA-dependent
recombination involves a four-stranded
Holliday intermediate which can resolve in
two ways. The integrity of DNA containing
unrepaired lesions can be maintained
during replication by homologous
recombination.
 Diploid eukaryotes: crossing over
1. Homologous chromosomes line up in meiosis (when)
2. The nonsister chromatids exchange equivalent sections (what)

back
 Haploid prokaryotes

Recombination between the two homologous DNA duplex (where)

• partially duplicated DNA of the chromosome
• between chromosomal DNA and “foreign” DNA

How: Holliday model

1. Nicks made near Chi

(GCTGGTGG) sites
by a nuclease.

2. RecA binds to ssDNA

to form RecA-ssDNA.

back
Haploid prokaryotes

3. RecA-ssDNA filaments search the opposite DNA duplex for

corresponding sequence (invasion).

4. Seal the nicks and form a four-branched Holliday structure

5. Branch migration & resolving Holliday junction

back
Haploid prokaryotes

Resolving
Holliday junction

back
 Mechanism of homologous recombination
2 pieces of ds DNA can pair through homologous
sequences
 Crossing over occurs between a single strand of
each ds molecule
 The Holliday junction is formed

 Breaking of the DNA strands can resolve the

Holliday structure in 2 ways
DNA repair in replication fork

a. Replication encounter a DNA lesion

b. Skip the lesion & re-initiate on the
side of the lesion
c. Fill the daughter strand gap by
replacing it with the corresponding
section from the parental sister
strand
d. post-replication repair of the left
lesion

back
 Site-specific recombination
1. Exchange of non-homologous but specific pieces of DNA
(what)
2. Mediated by proteins that recognize specific DNA
sequences. (how)

Example 1: bacteriophage l inserts its genome into a specific site on

the E. coli Chromosome

Example 2: Generation of antibody diversity in eukaryotes

back
 Site-specific recombination:
bacteriophage l insertion

1. l-encoded integrase (Int): makes staggered cuts in the specific sites

2. Int and IHF (integration host factor encoded by bacteria):
recombination and insertion
3. l-encoded excisionase (XIS): excision of the phage DNA

back
Site-specific recombination:
Antibody diversity

H and L are all encoded by three

gene segments: V, D, J

V D J

Two heavy 250 15 5

chains (H)
Two light 250 4
chains (L)

Enormous number (>108) of different H and L gene

sequences can be produced by such a recombination
back
One antibody example produced by site-specific recombination

back
Transposition
 transposable DNA elements (transposons) are
parasitic DNA sequences that colonize genomes and
can spread within them.
 they often disrupt the function or alter the
regulation of existing genes. On occasion, they can
create novel genes through fusions between
transposon and an existing gene
 Transposition

1. Requires no homology between sequences nor site-specific

2. Relatively inefficient
3. Require Transposase encoded by the transposon

transposons examples:
E. coli:
1. IS elements/, The insertion sequence (IS) consists of about
700–1500 base pairs (bp), containing a transposase gene and is
flanked by inverted repeats at both ends. The IS inserts itself at
the target site (The host DNA containing a of about 4–10 base
pairs) by means of the transposase activity

back
2. Tn transposon may contain other genes
such as for antibiotic resistance like b-
lactamase (penicillin resistance)and have
direct or inverted repeats at either end.
 Directrepeats are identical or closely related
sequences oriented in the same direction. Inverted
repeats are oriented in opposite directions.
Eukaryotic transposons, retrotransposons:

 Transposition of retroelements encodes

protein similar to RT (reverse
transcriptase)
 Retrotransposition requires synthesis of an

RNA copy of the inserted retroelement.

followed by reverse transcription up to
the polyadenylation sequence in the 3’
long terminal repeat (3 LTR).
1. Endogenous retroviruses are sequences that
resemble retroviruses but cannot infect new cells
and are restricted to one genome.
2. Nonviral retrotransposons lack LTRs and usually
other parts of retroviruses. Both types contain
reverse transcriptase and are therefore capable
of independent transposition.
3. Processed pseudogenes or retropseudogenes lack
reverse transcriptase and cannot transpose
independently
Transposition

back
Mutagenesis
Mutation
Replication fidelity
Physical mutagens
Chemical mutagens
Direct mutagenesis

DNA lesions
Oxidative damage
Alkylation
Bulky adducts
DNA repair
Photoreactivation
Alkyltransferase
Excision repair
Mismatch repair
Hereditary repair defects

Recombination
Homologous recombination
Site-specific recombination
Transposition