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DNA Recombination and Repair

DNA recombination:
General recombination (homologous recombination)
Site-specific recombination

DNA damage and repair:

DNA damage
DNA damage response
DNA repair pathways
Base excision repair
Nucleotide excision repair
Mismatch repair

DNA Recombination


DNA rearrangements between precisely corresponding sequences


1. General recombination (or homologous recombination)

2. Site-specific recombination

Key Terms of Recombination

Homologous recombination(generalized recombination) involves a

reciprocal exchange of sequences of DNA, e.g. between two chromosomes that
carry the same genetic loci.

Site-specific recombination(Specialized recombination) occurs between

two specific (not necessarily homologous) sequences, as in phage
integration/excision or resolution of cointegrate structures during transposition.

Transposition refers to the movement of a transposon to a new site in the


Homologous Recombination

Key Concepts
• Occurs at the “four strand” stage of
meiosis during spermatogenesis or
• Can occur anywhere in the homologous
nucleotide sequences of the two
participating strands
• Creates a heteroduplex region that can
be thousands of base pairs long
• No nucleotide sequences altered at the
site of exchange

Recombination Occurs at the Prophase of Meiosis

Recombination occurs during the first meiotic

prophase of meiosis. This Figure compares
the visible progress of chromosomes through
the five stages of meiotic prophase with the
molecular interactions that are involved in
exchanging material between two of the four
duplex DNAs.

Synaptonemal Complex Links Homolog Pairs

Key Terms
•The synaptonemal complex describes the
morphological structure of synapsed chromosomes.
•An axial element is a proteinaceous structure around
which the chromosomes condense at the start of
•A lateral element is a structure in the synaptonemal
complex. It is an axial element that is aligned with the
axial elements of other chromosomes.
•The central element is a structure that lies in the
middle of the synaptonemal complex, along which the
lateral elements of homologous chromosomes align.

Key Concepts
•During the early part of meiosis, homologous
chromosomes are paired in the synaptonemal complex.
•The mass of chromatin of each homologue is separated
from the other by a proteinaceous complex.

Recombination in Meiosis

Key Concepts
• Recombination is initiated by making a double-strand break in one (recipient) DNA duplex.
• Exonuclease action generates 3’ single-stranded ends that invade the other (donor) duplex.
• New DNA synthesis replaces the material that has been degraded.
• This generates a recombinant joint molecule in which the two DNA duplexes are connected
by heteroduplex DNA.

Spo11 Creates DSBs

Key Concepts
•Double-strand breaks that initiate recombination
occur before the synaptonemal complex forms.
•If recombination is blocked, the synaptonemal
complex cannot form.
•Spo11 is homologous to the catalytic subunits of a
family of type II topoisomerases.
•Spo11 interacts reversibly with DNA; the break is
converted into a permanent structure by an
interaction with another protein that dissociates the
Spo11 complex.
•Removal of Spo11 is followed by nuclease action.
•At least 9 other proteins are required to process
the double-strand breaks. One group of proteins is
required to convert the double-strand breaks into
protruding 3’–OH single-stranded ends. Another
group then enables the single-stranded ends to
invade homologous duplex DNA.

Role of RecBCD in Recombination
Key Concepts
•The RecBCD complex has nuclease and helicases

•It binds to DNA downstream of a chi sequence, unwinds

the duplex, and degrades one strand from 3’–5’ as it
moves to the chi site.

•The chi site triggers loss of the RecD subunit and

nuclease activity.

Chi Sequence


RecA catalyzes Strand Exchange

Key Concepts
RecA forms filaments with single-stranded or duplex DNA and catalyzes the ability of a single-
stranded DNA with a free 3’ to displace its counterpart in a DNA duplex.

RecA Creates Recombination Intermediates

RecA-mediated strand exchange between

partially duplex and entirely duplex DNA
generates a joint molecule with the same
structure as a recombination intermediate.

Role of RuvABC in Recombination

Key Concepts
•The Ruv complex acts on recombinant
•RuvA (27 kDa) recognizes the structure
of the junction.
•RuvB (37 kDa) is a helicase that
catalyzes branch migration.
•RuvC (19 kDa) cleaves junctions to
generate recombination intermediates.

Holliday Junction

Resolution of Holliday Junction

The continuous strands of the stacked­X structure (a) which form the wide angles in the 
unfolded RuvC­junction complex (b) are indicated by asterisks. The site of incision are 
indicated by scissors. The products of resolution (C) can be repaired by DNA ligase.

RecA Homologs in Eukaryotes

Rad51: catalyzes a synaptic reaction between a single strand DNA

and a DNA double helix.

Brca1 and Brca2: interact with Rad51 and facilitate strand


Recombination Can Lead to Gene Conversion

Key Terms
•Postmeiotic segregation describes the
segregation of two strands of a duplex DNA that bear
different information (created by heteroduplex
formation during meiosis) when a subsequent
replication allows the strands to separate.
•Gene conversion is the alteration of one strand of
a heteroduplex DNA to make it complementary with
the other strand at any position(s) where there were
mispaired bases.

Key Concepts
•Heteroduplex DNA that is created by recombination
can have mismatched sequences where the
recombining alleles are not identical.
•Repair systems may remove mismatches by
changing one of the strands so its sequence is
complementary to the other.

Recombination Functions as a DNA Repair Mechanism

DNA at the break site (a) is resected to expose

single-stranded (ss)DNA (b). The resulting ssDNA
becomes coated by the single-strand-binding
protein, replication protein A (RPA), which in turn
functions as a target for the binding of RAD52. As
RAD51 interacts with RAD52, RAD51 can gain
access to the ssDNA–RPA complex. (c) After
assembly of an active nucleoprotein filament on
the resected ssDNA tail of the first end, the
complex pairs with homologous double-stranded
(ds)DNA and strand exchange takes place. (c) In
the next stage, RAD51 and/or RAD52 promote the
capture of the second-end ssDNA tail. (d and e)
The two invading ends function as primers for
DNA resynthesis. (f) Finally, the DNA crossovers
are resolved to allow the repaired duplexes to

Double­Strand Break and 
Recombination Repair

Double-strand breaks are repaired by

either homologous recombination,
which in eukaryotes depends on the
Rad51-family proteinsorthologs of the
bacterial RecA recombinase, or by
nonhomologous end-joining mediated
by the DNA-PK complex. A key
intermediate in homologous
recombination is the Holliday
intermediate, in which the two
recombining duplexes are joined
covalently by single-strand crossovers.
Resolvases such as MUS81·MMS4
cleave the Holliday junctions to
separate the two duplexes. In the
single-strand annealing (SSA)
mechanism, the duplex is digested by
a 5' to 3' exonuclease to uncover
microhomology regions that promote
pairing, trimming, and ligation. BRCA1
and BRCA2 are also involved in
homologous recombination, but their
precise roles are unclear.

• Homologous recombination allows large section of the DNA to
move from one chromosome to another; occurs only between
homologous DNA molecules.

• Homologous recombination begins with a double-strand break

that is created by Spo11 and processed by RecBCD to expose a
single-stranded DNA end;

• Synapsis is catalyzed by RecA protein, and leads to the

formation of a Holliday junction;

• Resolution of the Holliday junction is conducted by RuvABC.

• Homologous recombination can lead to gene conversion and

repair double strand DNA breaks.

DNA Recombination and Repair

DNA recombination:
General recombination (homologous recombination)
Site-specific recombination

DNA repair:
DNA damage
DNA damage response
DNA repair pathways
Base excision repair
Nucleotide excision repair
Mismatch repair

Site­Specific Recombination

• Site-specific recombination, also called specialized recombination,

can alter gene order and add new information to the genome.

• Site-specific recombination moves specialized nucleotide sequences,

called mobile genetic elements, between nonhomologous sites within a

• Site-specific recombination occurs by two different mechanisms:

1. Transpositional site-specific recombination
2. Conservative site-specific recombination


Key Terms

Transposons (also called transposable elements):

Discrete sequences in genome that are mobile.

An enzyme encoded by the transposon and
disconnecting the transposon from one piece of DNA and
inserting it into a new target DNA site.

Classes of Transposable Elements

DNA­Only Transposon

Cut­and­paste transposition

Retroviral­Like Retrotransposition

Mechanism of Integrase

Key Concepts
•Integrases are related to topoisomerases, and the
recombination reaction resembles topoisomerase action
except that nicked strands from different duplexes are
sealed together.

•The reaction conserves energy by using a catalytic

tyrosine in the enzyme to break a phosphodiester bond
and link to the broken 3’ end.

•Two enzyme units bind to each recombination site and

the two dimers synapse to form a complex in which the
transfer reactions occur.

Non­retroviral retrotransposition

Conservative Site­Specific Recombination

Breakage and joining occur at two specific sites, one on each

participating DNA molecules. Depending on the orientation of the
two sites, DNA integration, excision, or inversion can occur.


Mobile genetic elements (transposons and viruses) can move from one
position in the genome to another by either a transpositional or a
conservative site-specificRecombination process

Site-specific recombination occurs randomly at a very low frequency.

There are three classes of transposons:

DNA-only transposons
Retroviral-like retrotransposons
Nonretroviral retrotransposons

DNA Damage and Repair
DNA damage and repair:
DNA damage
DNA damage response
DNA repair pathways and their relationship with cancers
Base excision repair
Nucleotide excision repair
Mismatch repair

Types of DNA Damage

DNA lesions and structures that elicit DNA response reactions. Some of the
base backbone lesions and noncanonical DNA structures that elicit DNA
response reactions are shown. O6MeGua indicates O6-methyl-
deoxyguanosine, T<>T indicates a cyclobutane thymine dimer, and the
cross-link shown is cisplatin G-G interstrand cross-link.
Endogenous DNA Damage

Deamination of cytosine to uracil can

spontaneously occurs to creates a U·G

Depurination removes a base from DNA,

blocking replication and transcription.

Mismatch Derived from Normal DNA Metabolism

5’ 3’




DNA replication errors DNA recombination

Exogenous DNA Damage
Ultraviolet irradiation introduces covalent
bonds between two adjacent thymine
bases and causes formation of intrastrand
pyrimidine dimer. The dimer blocks
replication and transcription.

Methylation of a base distorts the double

helix and causes mispairing at replication.

DNA Damage and Repair in Cancer

DNA damage and repair:

DNA damage
DNA damage response
DNA repair pathways and their relationship with cancers
Direct reverse
Base excision repair
Nucleotide excision repair
Mismatch repair
Double strand break repair

DNA Damage Response Pathways

DNA damage response reactions in mammalian cells. The four responses (DNA
repair, transcriptional response, DNA damage checkpoints, and apoptosis) may
function independently, but frequently a protein primarily involved in one response
may participate in other responses.

SOS Response in E. coli

Key Terms
•An SOS response in E. coli describes the
coordinate induction of many enzymes (more than
20), including repair activities, in response to
irradiation or other damage to DNA; results from
activation of protease activity by RecA to cleave
LexA repressor.

•The SOS box is the DNA sequence (operator) of

~20 bp recognized by LexA repressor protein.

Key Concepts
•Damage to DNA causes RecA to trigger the SOS
response, consisting of genes coding for many
repair enzymes.
•RecA activates the autocleavage activity of LexA.
•LexA represses the SOS system; its autocleavage
activates target gene expression.

DNA Damage Checkpoint 
DNA damage Checkpoints are
biochemical pathways that delay or
arrest cell cycle progression in
response to DNA damage. Like other
signal transduction pathways, the DNA
damage checkpoint has three types of
components: sensors, signal
transducers, and effectors. The
damage is detected by sensors that,
with the aid of mediators, transduce
the signal to transducers. The
transducers, in turn, activate or
inactivate other proteins (effectors)
that directly participate in inhibiting
the G1/S transition, S-phase
progression, or the G2/M transition.

Sensors: ATM and ATR Proteins
• ATM (ataxia telangiectesia mutated) is a 350-kDa protein with serine- and
threonine-specific protein kinase activity, it belongs to the phosphoinositide 3-
kinase-like kinase (PIKK). Upon exposure of cells to ionizing radiation (IR),
ATM phosphorylates many proteins, including p53, Chk1, NBS1, BRCA1, and

• Mutations of ATM cause ataxia-telangiectesia (A-T) in humans, a disease

characterized by cerebellar degeneration, immunodeficiency, genome
instability, clinical radio-sensitivity, and cancer predisposition.

• ATR (ATM-related) gene encodes for a protein of 303-kDa with protein kinase
activity. Mutations in ATR are associated with the human autosomal recessive
disorder Seckel syndrome, which shares features in common with A-T. In
contrast to ATM, ATR is activated by UV light rather than by IR.

• ATRIP (ATR interacting protein) is a 86-kDa protein that interacts with ATR.
ATRIP may confer some specificity for binding of the ATR-ATRIP complex to RPA
coated DNA rather than naked DNA.

Sensors: Rad17­RFC and the 9­1­1 Complex

• Rad17-RFC complex is a checkpoint-specific structure homolg of the

replication factor C (RFC). RFC is a heteropentamer composed of
p36, p37, p38, and p140 subunits. In Rad17-RFC, the p140 subunit is
replaced by the 75-kDa Rad17 protein.

• The 9-1-1 (Rad9-Rad1-Hus1) is a checkpoint counterpart of PCNA, a

homotrimer with a ring-like structure. Although 9-1-1 has little
sequence homology to PCNA, the complex forms a heterotrimeric ring
structure with a 2-nm hole that is very similar to that of PCNA.

• Rad17-RFC is a DNA-stimulated ATPase that binds both to DNA and

the 9-1-1 complex. Rad17-RFC recruits 9-1-1 complex to damaged
DNA in a ATP-dependent manner. Phosphorylation of Rad17 by ATR
upon DNA damage is necessary for the DNA damage checkpoint


• Mediators simultaneously associate with damage sensors and/or

signal transducers at certain phases of the cell cycle and as a
consequence help to provide signal transduction specificity.

• Examples: the p53 binding protein, 53BP1b, the topoisomerase

binding protein, TopBP1, and the mediator of DNA damage
checkpoint 1, MDC1 interact with damage sensor such as ATM, repair
proteins such as BRCA1 and Mre11/Rad50/Nbs1, signal transducers
such as Chk2, and even effcetor molecules such as p53.

Signal Transducers

• There are two signal transducers, Chk1 and Chk2. These S/T
kinases play a strict signal transduction function in cell cycle
regulation and checkpoint responses.

• In mammalian cells, DSB signal sensed by ATM is transduced by Chk2,

and the UV-damage signal sensed by ATR is transduced by Chk1.
However, there is some overlap between the functions of these two

• Knockout mouse studies indicate that Chk1 (-/-) cause embryonic

lethality in mice, whereas Chk2 (-/-) mice are viable and appear to
exhibit near-normal checkpoint response.


• In humans, three phosphotyrosine phosphatases, Cdc25A, -B, and –

C, act as effectors for the DNA damage checkpoint response. These
proteins dephosphorylate the cyclin-dependent kinases that act on
proteins directly involved in cell-cycle transitions.

• Phosphorylation of Cdc25 proteins by the checkpoint kinases

inactivates the proteins by excluding them from the nucleus, by
causing proteolytic degradation. Or both.

• Unphosphorylated Cdc25 proteins promote the G1/S transition by

dephosphorylating Cdk2 and promote the G2/M transition by
dephosphorylating Cdc2 phosphotyrosine.

G1/S Checkpoint

DNA damage is sensed by ATM after double-

strand breaks or by ATR, Rad17-RFC, and
the 9-1-1 complex after UV-damage.
ATM/ATR phosphorylates Rad17, Rad9, p53,
and Chk1/Chk2 that in turn phosphorylates
Cdc25A, causing its inactivation by nuclear
exclusion and ubiquitin-mediated
degradation. Phosphorylated and inactivated
Cdk2 accumulates and cannot phosphorylate
Cdc45 to initiate replication. Maintenance of
the G1/S arrest is achieved by p53, which is
phosphorylated on Ser15 by ATM/ATR and
on Ser20 by Chk1/Chk2. Phosphorylated p53
induces p21WAF-1/Cip1 transcription, and p21WAF-
binds to the Cdk4/CycD complex, thus
preventing it from phosphorylating Rb, which
is necessary for the release of the E2F
transcription factor and subsequent
transcription of S-phase genes. p21 WAF-1/Cip1
also binds to and inactivates the Cdk2/CycE
complex, thus securing the maintenance of
the G1/S checkpoint.

ATM­Regulated S­Phase 

In response to double-strandbreaks
induced by ionizing radiation, ATM
triggers two cooperating parallel
cascades to inhibit replicative DNA
synthesis. ATM, through the
intermediacy of MDC1, H2AX, and
53BP1, phosphorylates Chk2 on Thr68 to
induce ubiquitin-mediated degradation
of Cdc25A phosphatase. The degradation
locks the S phase-promoting Cyclin
E/Cdk2 in its inactive, phosphorylated
form and prevents the loading of Cdc45
on the replication origin. ATM also
initiates a second pathway by
phosphorylating NBS1 of the M/R/N
complex, as well as SMC1, BRCA1, and

ATR­Regulated S­Phase 

The ATR-ATRIP complex, Rad17-

RFC, the 9-1-1 complex, and
Claspin are independently
recruited to RPA(A)-coated single-
stranded regions of the stalled
replication fork. ATR then
phosphorylates Chk1 and other
substrates, and activated Chk1
phosphorylates Cdc25A, which
leads to inactivation of
Cdk2/Cyclin E complex. Single-
strand DNA gaps can also be
sensed by ATR and following ATR
activation, origin firing and
consequently DNA replication is
inhibited through downregulation
of Cdc7/Dbf4 protein kinase
DNA Repair

DNA damage and repair:

DNA damage
DNA damage response
DNA repair pathways
Direct reverse
Base excision repair
Nucleotide excision repair
Mismatch repair

Direct Repair of Photodimer
Direct repair by photoreactivation.
Photolyase binds to DNA containing a
pyrimidine dimer in a light-
independent reaction and flips the
dimer out into the active site pocket.
Catalysis is initiated by light. The
photoantenna cofactor,
methenyltetrahydrofolate (5,10-
MTHF), absorbs a photon and
transfers the excitation energy to the
catalytic cofactor, FADH-. Then, the
excited state FADH- transfers an
electron to the pyrimidine dimer,
splitting the dimer into two
pyrimidines. The electron returns to
the flavin radical to regenerate FADH-,
and the enzyme dissociates from the
repaired DNA.

DNA Repair

DNA damage and repair:

DNA damage
DNA damage response
DNA repair pathways
Direct reverse
Base excision repair
Nucleotide excision repair
Mismatch repair
Double strand break repair

Base Excision Repair

•Excision repair that is initiated by DNA

glycosylase is called base excision repair
(BER), because the damaged moieties are
excised as free bases.

•DNA glycosylases are key enzymes

required in BER, and they remove the
damaged or mismatched base from DNA by
cleaving the bond between the base and the

Base Excision Repair

DNA glycosylase removes the damaged

base as a free base, generating another
DNA damage called apurinic or
apyrimidinic (AP) site. The removal of
an AP site is initiated by a second class of
BER enzymes called AP endonucleases,
which specifically recognize AP site and
produce incisions or nicks in duplex DNA
by hydrolyzing the phosphodiester bonds
immediately 5’ the AP site, resulting in a
5’ terminal deoxyribose-phosphate. An
exonuclease called DNA
deoxyribophosphodiesterase (dRpase)
removes the sugar moiety. The single
nucleotide gap can be filled by DNA

Uracil­DNA Gylycosylase

Uracil-DNA glycosylase specifically removes uracil from DNA by

hydrolyzing the N-glycosylic bond linking the base to the sugar-
phosphate backbone.

DNA Glycosylases
Enzyme Substrate

Uracil-DNA glycosylase Uracil

5’-mC-DNA glycosylase 5-methylcytocine

Thymine-DNA glycosylase G-T mispair

MutY G-A mispair

3-mA-DNA glycosylase 3-methyladenine

FaPy-DNA glycosylase Formamidopyrimidine or 8-ox-G

PD-DNA glycosylase Pyrimidine dimer

Glycosylase Associated with Lyase Activity

Some glycosylases also contain lyase

activity. Glycosylase hydrolyzes the bond
between base and deoxyribose (using
H20), but a lyase takes the reaction
further by opening the sugar ring using an
amino (NH2) group to attack the
deoxyribose ring.

AP Endonuclease and Lyase

P en


AP (apurinic or apyrimidinic) endonucleases

cleave hydrolytically 5’ to AP site to yield
5’-deoxyribose-5-phosphate (colored) and 3’-OH
nucleotide residues. AP lyases cleave by β-
elimination to yield 5’-terminal deoxynucleoside-
5’-phosphate residues and 3’ terminal α, β-
unsaturated aldehydes (colored).

Mechanism of Base Excision Repair
A damaged base is removed by a DNA
glycosylase to generate an AP site.
Depending on the initial events in base
removal, the repair patch may be a single
nucleotide (short patch) or 2-10 nucleotides
(long patch). When the base damage is
removed by a glycosylase/AP lyase that
cleaves the phosphodiester bond 3' to the AP
site, APE1 endonuclease cleaves the 5' bond
to the site and recruits Polβ to fill in a 1-nt
gap that is ligated by Lig3/XRCC1 complex.
When the AP site is generated by hydrolytic
glycosylases or by spontaneous hydrolysis,
repair usually proceeds through the long-
patch pathway. APE1 cleaves the 5'
phosphodiester bond, and the RFC/PCNA-Polδ
/ε complex carries out repair synthesis and
nick translation, displacing several
nucleotides. The flap structure is cleaved off
by FEN1 endonuclease and the long-repair
patch is ligated by Ligase 1.
DNA Damage and Repair

DNA damage and repair:

DNA damage
DNA damage response
DNA repair pathways
Direct reverse
Base excision repair
Nucleotide excision repair
Mismatch repair
Double strand break repair

Nucleotide Excision Repair in E. coli

Nucleotide excision repair (NER) removes

and replaces a stretch of DNA that includes
the damaged base(s).

In E. coli, the UvrABC system is

responsible for NER.

UvrABC Endonuclease
103 kDa
DNA-independent ATPase
DNA-damage binding activity
Forms a dimer with UvrB
76 kDa
Endonuclease activity
Forms a dimer with UvrA

68 kDa
Endonuclease activity
Interacts with UvrB

Damage Recognition by UvrAB

A (UvrA)2(UvrB)1 complex forms (B

and C) and initially binds to DNA at a
site removed from the damage (D).
The protein complex tracks along the
DNA using a DNA helicase activity until
it encounter the damage. UvrA protein
dissociates from the complex, leaving
a stable UvrB-DNA complex (F). This is
associated with bending and kinking of
the DNA.

Incision of the Damaged Nucleotides by UvrBC

Following the formation of a stable

UvrB-damaged DNA complex (A and B),
UvrC protein binds at the site (C) and
induces a conformational change which
enables bound UvrB protein to nick the
DNA 4 nucleotides 3’ to the site of
damage (D) (shown as a pyrimidine
dimer). This reaction requires the
binding of ATP (or ATP[γS]) by UvrB
protein, but no ATP hydrolysis occurs at
this step. Following the 3’incision, UvrC
protein catalyzes nicking of the DNA 7
nucleotides 5’ to the dimer(E).

Repair Synthesis

DNA helicase II (UvrD) is required

for the release of an oligonucleotide
fragment following bimodal incision
and for the replacement of UvrC
protein (A and B). UvrB protein
remains bound to the gapped DNA
during the excision reaction and is
released during the repair synthesis
reaction catalyzed by Pol I (C).
DNA ligation completes the
nucleotide excision repair reaction.

The UvrABC System

The uvr system of NER includes three genes,

uvrA,B,C, that code for the components of a repair
endonuclease. First, a UvrAB combination recognizes
DNA damage, e.g., pyrimidine dimers and other
bulky lesions. Then UvrA dissociates (this requires
ATP), and UvrC joins UvrB. The UvrBC combination
makes an incision on each side, one 7 nucleotides
from the 5’ side of the damaged site, and the other
3-4 nucleotides away from the 3’ side. This also
requires ATP. UvrD is a helicase that helps to unwind
the DNA to allow release of the single strand between
the two cuts. The enzyme involved in the repair
synthesis is DNA polymerase I (although DNA
polymerases II and III can substitute for it).

Defects in NER Predispose to Cancer

Clinical features of xeroderma

pigmentosum (XP). Upper, The disease
typically presents with severe pigmentary
disturbances on the sun-exposed areas of
the skin. Lower, These clinical features can
occur at a very early age.

NER in Human Cells

There are 8 xeroderma pigmentosum

(XP) genes, called XP complementation
groups (from XPA to XPG, and XPV)
DNA damage is recognized by the XPC-
hHR23B protein (A). An open bubble
structure is then formed around a lesion in a
reaction that uses the ATP-dependent
helicase activities of XPB and XPD (two of
the subunits of TFIIH) and also involves XPA
and RPA (B). Formation of this open complex
creates specific sites for cutting on the 3' side
by the XPG nuclease and then on the 5' side
by the ERCC1-XPF nuclease(C). After a
24- to 32-residue oligonucleotide is released,
the gap is filled in by PCNA-dependent POL
ε or δ and sealed by a DNA ligase,
presumably LIG1(D).

Mechanism of Nucleotide 
Excision repair

The DNA damage is recognized by the cooperative

binding of RPA, XPA, and XPC-TFIIH, which assemble
at the damage site in a random order. The four repair
factors form a complex at the binding site, and if the
binding site is damage-free, ATP hydrolysis by the
XPB and XPD helicases dissociates the complex
(kinetic proofreading). If the site contains a lesion,
ATP hydrolysis unwinds the duplex by about 25 bp
around the lesion, making a stable preincision
complex 1 (PIC1) at the damage site. XPG then
replaces XPC in the complex to form a more stable
preincision complex 2 (PIC2). Finally, XPF·ERCC1 is
recruited to the damage site to form preincision
complex 3 (PIC3). The damaged strand is incised at
the 6th ± 3 phosphodiester bond, 3' to the damage
by XPG, and the 20th ± 5 phosphodiester bond 5' to
the damage by XPF·ERCC1. The resulting 24-32
oligomer is released, and the gap is filled by Polδ/ε
with the aid of replication accessory proteins PCNA
and RFC.

Xeroderma Pigmentosum Complementation Group 


XPV (variant) A DNA polymerase (Polη) involved in

Translesion Synthesis (TLS)

5’ A A
3’ T T

DNA Damage and Repair

DNA damage and repair:

DNA damage
DNA damage response
DNA repair pathways
Direct reverse
Base excision repair
Nucleotide excision repair
Mismatch repair

DNA Mismatch Repair

• Mismatch repair in E. coli

• Mismatch repair in mammalians

• Mismatch repair deficiency and cancer

• Mismatch repair -mediated apoptosis

What is DNA Mismatch?

What is mismatch?
Non­Watson­Crick base pairs

Two classes of mismatches
Base­base mismatch
G­T, A­C, A­A, A­G, G­G, C­C, C­T, T­T

Insertion­deletion (unpaired) mismatch

Key MMR Components

97 kDa
Mismatch recognition protein
ATPase activity

70 kDa
ATPase activity

23 kDa
Endonuclease activity

Mismatch Repair in E. coli

DNA containing mismatched base partners

is repaired preferentially by excising the
strand that lacks the methylation. The
excision is quite extensive; mismatches can
be repaired preferentially for >1 kb around
a GATC site. The result is that the newly
synthesized strand is corrected to the
sequence of the parental strand.

In E. coli, three Mut proteins (MutS, MutL,

and MutH) are key components involved in
mismatch repair.

MutS, L, and H Proteins

MutS binds to the mismatch and is joined by

MutL and MutH. Using an unclear
mechanism, the repair machinery identifies
hemimethylated GATC sequence in the
presence of ATP. MutH (an endonuclease)
then cleaves the unmethylated strand at the
GATC site. This strand is then excised from
the GATC site to the mismatch site. The
excision can occur in either the 5’–3’ direction
(using RecJ or exonuclease VII) or in the
3’–5’ direction (using exonuclease I),
assisted by the helicase UvrD. The gap is
filled by DNA polymerase III in concert with
single-strand DNA binding protein (SSB).

Mismatch Repair in E. Coli
CH 3 CH 3


CH 3
MutL CH3

Pol III holoenzyme
MutH Helicase II
CH 3 SSB  CH 3

ExoI, ExoVII, 

Models for the Initiation of Mismatch repair
A, Translocation model. ATP reduces the
mismatch binding affinity of MutS dimer and
ATP hydrolysis drives bi-directional translocation
of MutS to form an α-like loop structure of DNA.
B, Molecular switch model. MutS is suggested to
be present in either an ADP-bound form or an
ATP-bound form. Binding of the ADP-bound
MutS to a mismatch stimulates the exchange of
ADP for ATP. The nucleotide switch results in a
conformational change of MutS and promotes
the ATP-MutS complex to diffuse along the DNA
helix. ATP is not hydrolyzed in this diffusible
complex. C, Transactivation model. Binding of
ATP enables MutS to release a homoduplex DNA
(upper) or to continue to bind a heteroduplex
DNA through a loosening interaction (lower). In
the presence of MutL, MutH, and the hemi-
methylated GATC sequence, the bound ATP
molecules allow the MutS-mismatch complex to
form a repair complex of the three proteins with
two DNA recognition sites and initiate mismatch
repair. The ATP bound by MutS is shown in
purple, and the ATP hydrolyzed by MutL during
the trans-activation is shown in orange.
Characteristic of MMR

• MutHLS-dependent

• Bi-directional process

• Strand specificity (repair occurs only on the newly

synthesized strand)

Crystal Structure of MutS

DNA Mismatch Repair

• Mismatch repair in E. coli

• Mismatch repair in humans

• Mismatch repair deficiency and cancer

• Mismatch repair -mediated apoptosis

Human Mut Homologs

E. coli Human
MutSα MutSβ
(MutS)2 MSH3

(MutL)2 MutLα PMS1 MutLβ

MutH ?

MMR Components in Humans
E. coli Human
MutS MutSαMutS
 β
MutL MutLα MutLβMutL
 γ
MutH ?
UvrD ?
ExoI, ExoVII, ExoX, RecJ ExoI (others?)
Pol III holoenzyme Pol δ (α, ε?)
DNA ligase ?
Human Mismatch Repair

The human MMR system can bi-directionally process all eight base-base mismatches
and 1-16 nt ID mispairs. The repair process in each case involves repair initiation,
excision, and resynthesis. Except for mismatch recognition, where hMutSα and hMutS
β are required to distinguish specific substrates as indicated, activities required for the
remaining steps of the reaction are believed to be the same for the processing of
base-base mismatches and ID mispairs.  
DNA Mismatch Repair

• Mismatch repair in E. coli

• Mismatch repair in mammalians

• Mismatch repair deficiency and cancer

• Mismatch repair -mediated apoptosis

Microsatellite and Its Instability

Key Terms
Simple repetitive DNA sequences are called microsatellite.
For example: (A)n
There are hundreds and thousands of microsatellite
sequences in our DNA, and the are located mostly in non-
coding regions.

Microsatellite instability (MSI):

Alterations in repeat numbers of microsatellite sequences
from one generation to another.

Microsatellite Instability in Cancer

Dinucleotide repeat polymorphisms in normal (N) and tumor (T) tissue from patients
with hereditary non-polyposis colorectal cancer (HNPCC) (A and B) and patients with
sporadic colorectal cancer (C and D). Genomic DNAs were amplified by PCR using
microsatellite markers D2S123 abd D10S197, and the products were separated in 6%
polyacrylamide gels.

Science, Vol, 260, 812-816 (1993)

Mechanism of MSI

Loop out in the template strand


Loop out in the primer strand


MMR Genes and Colorectal Cancer

HNPCCa Sporadic
Population incidence ~1 in 500 1 in 20
Microsatellite instability >90% of kindreds 13%
MMR gene mutations 70% ~65% of CRC with MSI

MSH2 45% 50%

MSH3 0%
MSH6 0.5%

MLH1b 48% 50%

PMS1 One family
PMS2 5%
MLH3 ?
Hereditary non-polyposis colorectal cancer.
Hypermethylation of the MLH1 gene silences expression of MLH1
and leads to a defective MMR system.
Detection of MLH1 Promoter Hypermethylation

Sodium bisulfite


7 8 17 10 15 16 L E M H2O

Hypermethylation in sporadic breast cancers. DNA from breast tumors were tretaed
with sodiumn bisulfite. The C region of the hMLH1 promoter was amplified by PCR.
M, methylation specific primers; U, unmethylation-specific primers.

Oncogene, Vol. 21, 5696-5703 (2002)

Detecting Methylation by Restriction Enzymes

Sensitive to
HpaII Yes No
MspI Yes Yes

5’ GCGC 3’ 5’ G mC G C 3’
3’ CGCG 5’ 3’ C G mC G 5’
HpaII or Msp1 HpaII Msp1

PCR products No Yes No

MLH1 Hypermethylation in Sporadic Cancers

Analysis of methylation status of the hMLH1 promoter. Shown is the product resulting from PCR
amplification of the hMLH1 promoter region before or after digestion. U, Undigested; H, digested
with HpaII; M, digested with MspI. The methylation status (+, methylated; -, unmethylated) of the
hMLH1 promoter is designated below each sample.

PNAS, Vol. 95, 8698-8702, 1998

Induction of hMLH1 Expression by 5-azacytidine

hMLH1 protein expression in MSI cancers.

Shown is a Western blot of hMLH1 protein
expression. The presence (+) or absence (-)
of MSI and the status of the coding region of
the hMLH1 gene (wt, wild type; mut, mutant)
are denoted below each lane.

Induction of hMLH1 protein expression

after 5-azacytidine (AzaC) treatment.
hMLH1 was detected by Western blot. U,
Cells untreated; T, cells treated with 5-

PNAS, Vol. 95, 8698-8702, (1998)

Germline Epimutation of MLH1 in Cancer

Epigenetic silencing can mimic genetic mutation by abolishing expression of

a gene. It was hypothesized that an epimutation could occur in any gene
as a germline event that predisposes to disease. Suter et al. looked for
examples in tumor suppressor genes in individuals with cancer, and found:
1. Two individuals with soma-wide, allele-specific and mosaic
hypermethylation of the DNA mismatch repair gene MLH1. Both individuals
lack evidence of genetic mutation in any mismatch repair gene but have
had multiple primary tumors that show mismatch repair deficiency, and
both meet clinical criteria for hereditary nonpolyposis colorectal cancer.
2. The epimutation was also present in spermatozoa of one of the
individuals, indicating a germline defect and the potential for transmission
to offspring.

Nature Genetics 36, 497 - 501 (2004)

DNA Mismatch Repair

• Mismatch repair in E. coli

• Mismatch repair in mammalians

• Mismatch repair deficiency and cancer

• Mismatch repair -mediated apoptosis

DNA Damage­Induced Apoptosis

MNNG - + - +

Cell line MMR Mutation

TK6 Proficient WT

MT1 Deficient MutSα

MutSα Binds to Damaged DNA

O6-methylguanine (MNNG, procarbazine, temozolomide)

Cisplatin adducts

8-Hydroxyguanine (oxidative damage)

Aminofluorene adducts

Adducts of polycyclic aromatic hydrocarbon (B[a]P, B[c]Phe)

UV dimer

Mechanism of MMR­Mediated Apoptosis

A, The futile repair cycle model. The adducted base

(solid circle) could pair with an appropriate base or lead
to a mispair during DNA replication. This abnormal
structure can be recognized by MutSα (MSH2-MSH6)
and provoke a strand-specific MMR reaction. However,
since mismatch repair (MMR) can be only targeted to
the newly synthesized strand (red line), the offending
adduct in the template strand (black line) cannot be
removed, and will initiate a new cycle of MMR upon
repair resynthesis. Such a futile repair cycle stimulates
an interaction between MMR proteins and apoptotic
transducers (e.g. ATM, c-Abl, p73, and p53) to
activate the apoptotic machinery. B, The blockage
model. The binding of MutSα/MutLα to a DNA adduct
in the replication fork and/or the unwound DNA helix
could block DNA transactions such as replication,
transcription, and repair. The blockage promotes MMR
proteins to interact with apoptotic transducers to switch
on apoptotic machinery.

Tumor Suppression of MMR
Wild­type cells MMR mutant cells

Biosynthetic errors Correction Hypermutable

DNA damage Apoptosis Survival with mutation

Consequence Mutation­free genome Tumorigenesis

Functions of MMR

• Repair function
Correcting heteroduplexes

• Apoptosis function
Eliminating damaged cells from body by promoting apoptosis


Defects in MMR are the genetic basis for certain types of cancer.
1. Genetic mutations, particularly in MSH2 and MLH1
2. Hypermethylation of MLH1 gene

MMR maintains genomic stability via at least two distinct pathways.

1. Repair function
• Relying on MutSLH and their homologs for repair initiation
• Occurring only in the newly synthesized strand
• Bi-directionality
2. Apoptosis function
• Recognizing a variety of DNA damage
• Initiating futile repair cycles to induce apoptosis