Chapter 30:

DNA Replication, Repair,

and Recombination
1. DNA Replication: An overview
2. Enzymes of Replication
3. Prokaryotic Replication
4. Eukaryotic Replication
5. Repair of DNA
6. Recombination and Mobile Genetic
Elements
7. DNA Methylation and Trinucleotide
Repeat Expansion
 DNA Replication

• DNA double strand -> template for duplication,

Replication
• Chemically similar to transcription
• As complex as translation but enzymes in only few
copies/cell

• Extremely accurate: 10-10 mistakes/base

• Extremely regulated: only once per cell division
Action of DNA polymerase

• Template
• dNTPs
• 5’ -> 3’ direction
• Semi-conservative
Replication of DNA
Unwinding of dsDNA:
- Rate in E. coli: 1000nt/sec
- 100rev/sec (10bp/turn)

Negative supercoils by:

DNA Gyrase
type II topoisomerase, ATP
 E. coli theta replication

• autoradiogramm
• branch point called “replication fork”
• unidirectional / bidirectional
• prokaryotes and bacteriophages have only one
origin of replication
 Unidirectional vs.
bidirectional θ replication

[3H]tymidine pulse-labelling
Semidiscontinuous DNA
replication

Okazaki fragments:
Discontinous ! 1000-2000nt in prokaryotes
100-200nt in eukaryotes
Joined by DNA ligase
Replication eye in Drosophila
melanogaster DNA
Priming of DNA synthesis
by short RNA segments

E. coli:
RNA Polymerase
Primase, rifampicin sensitive
Removal of RNA primers
2. Enzymes of replication

DNA Replication requires (in order of appearance):

1. DNA Topoisomerase
2. Helicases
3. ssDNA binding proteins
4. RNA primer synthesis
5. DNA polymerase
6. Enzyme to remove RNA primers
7. Link Okazaki fragments
E. coli DNA polymerase I in complex
with a dsDNA
Arthur Kornberg, 1957
DNA Polymerase I
5’->3’ synthesis
Processive, 20nt
Recognizes dNTP based
on base pairing
Right hand sructure

Editing activity:
3’->5’ exonuclease
5’->3’ exonuclease
(proofreading)
Fidelity 10-7

Klenow fragment
Lacks 5’->3’ exo, lacks N-
term.
 Nick translation as catalyzed
by Pol I

Used to radiolabel DNA probes for Southern/Northern

DNaseI, αP32dNTP
 Pol I functions to repair
DNA
E. coli, Pol I mutant are viable but sensitive to UV
and chemical mutagens

Essentisl physiological function of Pol I 5’->3’

exonuclease is to excise RNA primers, role in replication
 DNA Polymerase III

Pol III is replicase of E. coli

Holoenzyme consists of more than 10 subunits
β subunit confers processivity >5000nt
β subunit form a ring like sliding clamp
with 80Å diameter hole, sliding clamp/ β clamp
Properties of E. coli DNA
Polymerases
Components of E. coli DNA
Polymerase III Holoenzyme
β subunit of E. coli Pol III
holoenzyme
 Unwinding of DNA

3 proteins required to advance replication fork:

Helicase, DnaB, hexameric, ATP-dep., 5’->3’,AAA+
Strand separation, Rep helicase, dimer, ATP-dep.
ssDNA binding protein, prevent re-annealing, tetramer
Unwinding and Binding Proteins
of E. coli DNA Replication
 Active, rolling mechanism for
DNA unwinding by Rep helicase
 DNA ligase

Ligating single strand nicks

between Okazaki fragments

E. coli: NAD-dependent
T4 phage, ATP-dependent
blunt end ligation
 Primase
Synthesis of RNA primers fro Okazaki fragments:
5’->3’
In vitro 11nt ±1
 Prokaryotic Replication

Bacteriophages
Coliphages: M13, φX174

M13: 6408nt
ssDNA(+), circular
Replication->RF
Leading strand synthesis
 φX174 Replication
5386nt ssDNA circular
Replication more complex than M13
Requires primosome
Paradigm for lagging strand synthesis
6step process
a. coating
b. primosome assembly
c. migration
d. priming
e. Pol III extension
f. Pol I removes primers
g. ligation, supercoiling
Micrograph of a primosome
Proteins of the Primosomea
The rolling circle
mode of DNA
replication

a. Specific cut at + strand

b. Extension of + strand
c. Tandem-linked + strands
d. Separation by endonuclease
e. packaging

Rolling circle = Sigma replication

 φX174 (+) strand
replication by the looped
rolling circle mode

φX174 (+) strand synthesis

as model for leading strand
replication
1. Cut by gene A protein
2. Pol II extension
3. Cut + ligation
 The replication of E. coli DNA
Bidirectional, theta replication
leading and lagging strand synthesis occurs
on a common 900kD multisubunit particle:
the replisome -> loop of lagging strand

Initiation: at oriC, 245bp segment

The replication of E. coli DNA
 A model for DNA
replication initiation at
oriC

oriC, 245bp segment

Contains 5 DnaA boxes
Melting,
P1 Penicillium citrinum endodunclease
Specific for ssDNA
Prepriming complex (DnaB DnaC)6
Initiation of DNA replication is
strictly regulated
Only 1 replication/cell cycle
Doubling time 20min-10h
1000nt/sec
4.6 106bp genome
-> 40min/replication
-> multiforked
chromosomes
Sequestration of
hemimethylated oriC
 Electron micrograph of an intact and
supercoiled E. coli chromosome attached to
two fragments of the cell membrane
 Schematic diagram of the
clamp loading cycle
β clamp responsible for high processivity of Pol III
Must be “loaded” onto DNA by a clamp loader
ATP-dep. AAA+
 Termination of replication
Large 350 kb region in E. coli genome
Flanked by 7 nonpalindromic nearly identical termination
Sites
Replication fork counterclockwise passes through
TerG,F, B, and C but stops at TerA
Analogous for other direction
Ter act as valves
Ter-action requires binding of
Tus protein

Without Ter, collision of

replication forks terminates
 Fidelity of Replication
Complexity of replication (>20 proteins) important
for high fidelity:
T4 phage reversion 10-8 - 10-10

High accuracy due to:

1. Balanced dNTP levels
2. Polymerase reaction itself, pairing
3. 3’->5’ exonuclease of Pol I and Pol III
4. Repair systems -> see later
 Why only 5’->3’ synthesis ?

3’->5’ extension would require retention of 5’ triphosphate

This would be lost upon editing !
 Eukaryotic Replication
Remarkable degree of similarity to prok. replication
But linear chromosomes -> ends ?

Cell cycle regulation, can last 8h to > 100 days

Most variation in G1 phase/Go phase
Irreversible decision to proliferate is made in G1
Checkpoint
Controlled by cyclins and cyclin-dep. kinases

Best understood from yeast (budding, fission)

The eukaryotic cell cycle
 Eukaryotic cells contain
many polymerases
6 families:
A, E. coli Pol I, Pol γ (mitochondrial)
B, E. coli Pol II, Pol α, Pol δ
C, E. coli Pol III
D, X, Y

Pol δ, unlimited processivity when in

complex with PCNA, proliferating cell
nuclear antigen (systemic lupus
erythematosus), β clamp function
Properties of Some Animal
DNA Polymerases
Structure of PCNA
 Eukaryotic chromosomes consist
of numerous replicons

Multiple replication origins, every 3-300kb

Replication rate 50nt/sec, 20x slower than E. coli
But 60x more DNA
Replication would require 1 month
Clusters of 20-80 adjacent replicons
Not simultaneously, but ensure they initiate only once
 Assembly of the initiator
complex in 2 stages
To prevent multiple rounds of
initiation:
Assembly of pre-RC in G1 phase
(licensed)
Activation at S phase
Origin can “fire” only once
Origin = ARS (autonomously
replicating sequences)
Re-replication prevented by
Cdks and Geminin

ORC, origin recognition complex

Hexamer, Orc1-Orc6 (DnaA analog)
MCM, minichr. maintenance funct.
 Removal of RNA primers
2 enzymes:
RNase H1, removes most of the RNA leaving a
single 5’ ribonucleotide (H, hybrid)

Flap endonuclease-1 (FEN1) removes single single

5’ ribonucleotide
Mitochondrial DNA
is replicated in D-
loops

15kb circular genome

Leading strand synthesis precedes
lagging strand

Leading strand forms displacement

loop (D-loop)
 Reverse transcriptase
Retroviruses:
RNA containing eukaryotic viruses, e.g. HIV
Replicate from RNA genome
Copy RNA into DNA by Reverse Transcripase (RT)

Similar to Pol I, 5’->3’ synthesis of DNA from RNA

template, primed by host tRNA
RNA is degraded by RNase H
ssDNA directs dsDNA synthesis
dsDNA integration into host genome

RT: important tool for cDNA synthesis, oligo-dT primed

Reverse transcriptase
 Structure of HIV-1
reverse transcriptase
RT inhibitors
 Telomers and Telomerase

How are the ends of linear chromosomes replicated ?

Problem: no priming at 5’ of lagging strand possible without

shortening of the chromosome upon every replication

Telomer sequence: unusual, G-rich, 3’ overhang

(20-200bp)

Specialized enzyme: telomerase adds G-rich repeats

without teplate, is ribonucleoprotein, RNA acts as
template
Synthesis of telomeric DNA
by Tetrahymena telomerase
 Telomers must be capped
Without telomerase, chromosome would shorten 50-100nt
with every cell division

Exposed telomeric ssDNA must be protected by capping

with proteins, Pot1
 Telomere length correlates
with aging
Primary cells in culture die after 20-60 divisions

Such somatic cells have no telomerase activity ->

Telomers shorten with every division
Telomerase is active only in germ cells

Analysis of fibroblast from donors of different age:

No correlation with numbers of doublings in culture
But correlation of telomer length with numbers of doublings

Progeria: premature aging disease

patients have short telomers
 Cancer cells have active
telomerase

Why do somatic cells down regulate telomerase ?

Senescence may be a mechanism to protect from cancer

All immortal cells express telomerase

Telomeric DNA can dimerize
via G-quartets
Telomers form T-loops
 Repair of DNA

DNA is not inert

UV radiation, ionizing radiation, toxic chemicals,

oxidative metabolism can harm DNA

Spontaneous hydrolysis of 10’000 glycosidic bonds in

every cell every day....

Human genome 130 genes dedicated to DNA repair

Chemically similar in E. coli
 Chemical damage of DNA

Oxidation
Hydrolysis
Methylation
 Direct reversal of damage
Pyrimidine dimers are
split by photolyase:

UV (200-300nm) promtes
Formation of cyclobutyl
ring between adjacent thymine
-> intrastrand thymine dimer
 DNA photolyase
Photoreactive enzyme:
Absorbed light is transferred to FADH-
Electron used to split thymine dimer

Base flipping:
Often used to repair damaged DNA
 Excision repair

Cells have two types of excision repair:

1. Nucleotide excision repair, NER
repairs bulky lesions
2. Base excision repair, BER
repairs nonbulky lesions involving a
single base
 Excision repair (NER)
Found in all cells
Activated by helix distortion
Major defense in humans
(cigarette smoke, carcinogens)

16 subunits, 3 in bacteria

E. coli: UvrA, UvrB, UvrC

UvrABC endonuclease

1. Cleavage
2. Displacement, UvrD
3. Repair, Pol I
 NER diseases
Xenoderma pigmentosum
skin cells cannot repair UV damage
Individuals extremely sensitive to sun light
skin tumors risk 2000-fold elevated
cultured skin cells are defective in repairing
tymidine dimers
Cell fusion experiments: 8 complementation groups

Cockayne syndrome
light sensitive and neurological defects
demyelination-> oxidative damage in neurons
 Base excision repair
Single base repair:

1. DNA glycosidase->
Apurinic or apyrimidinic
(AP) site (abasic site)

2. Ribose cleaved by
AP endonuclease

3. Exonuclease

4. Filled by pol and ligase

Uracil in DNA would be highly
mutagenic
Why use thymine in DNA and uracil in RNA ?

Cytosine deaminates to uracil

If U in DNA: no way to discriminate whether

G-U mismatch is due to:
G-C -> deaminated to U
A-U

Since T is normal in DNA, every U is due to

deaminated C
 Mismatch repair
Replicational mispairing is repaired by
mismatch repair (MMR)

Defects result in hereditary nonpolyposis

colorectal cancer (HNPCC)

Must distinguish between correct and wrong base

In E. coli, possible due to hemimethylation
3 proteins, MutS, MutL, MutH
Mismatch repair
in E. coli

1. MutS binds mismatch as dimer

2. MutS-DNA recrutes MutL
3. MutS-MutL scan DNA for hemi-
Methylated GATC, recrute MutH
4. Cleavage of non-methylated strand
5. Strand separation by UvrD
6. Exonuclease
7. Fill Pol III
8. Ligate
 The SOS response
On heavy DNA damage, E. coli stops to grow and induces
DNA repair system, SOS system

SOS operon, recA, uvrA, uvrB repressed by LexA

RecA is ssDNA binding protein, induces cleavage of LexA

upon ssDNA binding -> release repression of SOS operon
Regulation of the SOS response in
E. coli
 SOS repair is error prone

If replisome encounters DNA lesion:

Stallment, relase Pol III core, collapse of replication fork

To resume: either SOS repair or recombination repair

Recombination repair: circumvents lesion and uses

homologous recombination to restore damaged site (->later)

In SOS repair, Pol III is replaced by bypass DNA

polymerase, Pol IV or Pol V
Error prone polymerases -> SOS response is mutagenic ->
Adaptation to difficult situation by generating diversity
 Double-strand break repair

Ionizing radiation and free radicals can induce double

strand breaks in DNA (DSB)
Also induced by some cellular processes, e.g. VDJ recomb.

2 ways to repair DSBs:

1. Recombination repair-> later
2. Nonhomologous end-joining (NHEJ)
involves DNA end binding protein Ku
Nonhomologous
end-joining
(NHEJ)
 Identification of
carcinogens
Many forms of cancers are caused by exposure to
certain chemical agents, carcinogens (man-made or natural)

Ames test assay for carcinogenicity

Salmonelle typhimurium
his- incubate with chemical -> rate of reversion
to his+ correlates with mutagenecity of tested
chemical
 The Ames test for
mutagenesis
Filter disc containing
Substance:

1. Zone lethal
2. Zone mutagenic
3. Zone spontaneous
reversion
 Recombination and mobile
genetic elements

Pairs of allelic genes may exchange chromosomal location

by genetic recombination via homologous recombination

Homologous recombination:
Exchange of homologous segments between two DNA
molecules

Bacteria, haploid, exchange via conjugation (mating) or

Transduction (viral)
 The Holliday model of
homologous recombination
1. ssDNA nick
2. Strand invasion
3. Branch migration
4. Holliday interm.
Chi structure
5. Resolution
 Homologous
recombination
between two circular
DNA duplexes

Results either in two circles

of the original sizes or in a
single composite circle
 Homologous recombination in E.
coli is catalyzed by RecA
RecA mutants have 104-fold lowe rate of recombination
RecA catalyzes ATP-dependent strand exchange
Binds DNA with 6.2 RecA monomers/turn
Electron microscopy–based image (of
an E. coli RecA–dsDNA–ATP filament
 Model for RecA-mediated
pairing and strand exchange
 RecA-catalyzed assimilation
of a single-stranded circle

Requires:
-free end (nick)
-homology at 5’
 Hypothetical model for the RecA-
mediated strand exchange reaction

Rad51 is eukaryotic homologue of RecA

 recBCD initiate recombination
by making single-strand nicks
Products of the SOS operon
Unwinding dsDNA
exonuclease

to Chi sequence GCTGGTGG

Every 5kb
Have elevated rate of
recombination

Requires free ds ends:

Transformation
Conjugation, Transduction
Replication fork collaps
RuvABC mediates branch migration and
the resolution of the Holliday junction
Branch migration is ATP-dependent, unidirectional
Mediated by SOS-induced proteins:
RuvB, ATP-dep. Pump, hexamer, AAA+
RuvA, binds Holliday junction, homotetramer
RuvC, exonuclease
 Recombination
repair
Transformation, transduction and
conjugation are rare events requi-
ring recombination

Frequent is collapse of replication

fork, 10times/euk cell cycle
-> Recombination Repair
1. Replication arrest at lesion
2. Fork regression, chicken foot
3. Fill by Pol I
4. Reverse branch migration
(RecG)
5. Replication restart
Note: lesion is not repaired
 Recombination
repair of a single-
strand nick
Replication fork encounters ss
nick:
1. Collapse
2. RecBCD + RecA invasion
3. Branch migration, RuvAB
4. Resolution, RuvC
-> nick has become 5’ end of
Okazaki fragment
 Recombination repair reconstitutes
doulbe-strand breaks
Homologous end-joining as
alternative to NHEJ
2 Holliday junctions inter-
mediate

1. Resection of DS ends
2. DNA dynthesis and ligation
3. Resolution of 2 Hol.j.
 Transposition and site-
specific recombination
1950 Barbara McClintock, varied pigmentation on maize
Due to the action of variable genetic elements, i.e.
non-Mendelian inheritance
20 years later, evidence for mobile genetic elements in
E. coli
Transposable elements, transposons in prokaryotes and
euk.
Each transposon encodes for a transposase that
catalyzes illegitimate recombination, because it requires
no homology between donor and acceptor
Transposition is mutagenic and dangerous, tightly
regulated: 10-5 to 10-7 events per cell division
 Prokaryotic transposons
3 Types:
1. Simplest, insertion sequences, IS Elements
<2000bp, transposase, flanked by short
inverted repeats, flanked by direct repeat
at insertion site, E. coli: 8 copies of IS1,
5 copies of IS2
Properties of Some Insertion
Elements
 Transposons (2)
3 Types:
2. More complex, carry additional genes, e.g. anti-
biotic resistance
Example, Tn3, 4957 bp
a. transposase, TnpA
b. Recombinase, TnpR
c. beta-lactamase, Ampicilin resistance
 Transposons (3)
3 Types:
3. Composite transposons
gene containing central region flanked by
IS-like modules that have the same or
inverted orientation
Generation of direct repeats of the target
sequence by transposon insertion
 Two modes for transposition
1. Direct or simple transposition -> transposon moves from
position A to position B

2. Replicative transposition -> transposon remains + new

copy at position B
Direct transposition
of Tn5 by a cut and
paste mechanism

1. Transposase binding
2. Dimerization
3. Synaptic complex
4. Target capture
5. Integration
Replicative transposition
A cointegrate
 Model for
transposition
via cointegrate
1. Pair of staggered ss cuts
2. Ligation of both ends at
integration site
forms replication fork
3. Replication forms
cointegrate
4. Site-specific recombination
cointegrate resolved
 γδ Resolvase
catalyzed site-
specific
recombination

Via double-strand DNA cleavage

Replicative transposons are responsible for
much genetic remodeling in prokaryotes
Transposons induce rearrangements in host genome
a) Inversion of genomic segment
b) Deletion of genomic segment
Mediate transfer of genetic material between species
 Phase variation is mediated by
site-specific Recombination
Salmonella typhimurium make 2 antigenetically distinct
versions of flagellin, H1 and H2
only one of the two is expressed
switch every 1000 cell generations, phase variation
may help evade host immune response

H2 is linked to rh1, that encodes a repressor for H1

Expression of H2-rh1 unit is controlled by a 995bp
segment that contains
1. Promoter for H2-rh1
2. Hin gene coding for Hin DNA invertase
3. Two closely related 26bp sites, hixL and hixR
 Mechanism of
phase variation in
Salmonella
 Cre-mediated site-specific
recombination

Many bacteriophages have two modes to propagate:

1.lytic, lysis of cells
2. Lysogeic, integration into host genome

Examples: Bacteriophage lambda, λ integrase

P1 bacteriophage, Cre recombinase
 The circularization of linear
bacteriophage P1 DNA

34bp LoxP site, palindromic except for central 8bp

Mechanism of Cre–loxP site-
specific recombination

Via 3’-PhosphoTyr intermediate

Structure of the Cre tetramer
complexed with loxP DNA
 Most transposition in eukaryotes
involve RNA intermediates
3% of the human genome consists of transposons
Many are fosils, i.e. sequence mutated to be inactive
Many ressemble retroviruses in sequence

Retroposons
Transposition via RNA intermediate, tanscription
dsDNA via reverse transcriptase, cDNA
Random integration by integrase

Retroviral genome flanked by LTR, long terminal repeats

(250-600bp)
3 polyproteins: gag (viral core)
pol (reverse transcriptase)
env (viral envelope)
Organization of retroviruses
and the Ty1 retrotransposon
 Non-viral
retroposons
Vertebrate genomes contain
Retroposons that lack LTRs
Non-viral retroposons,e.g.
LINEs, long intersoersed nuclear
elements, 1-7kn long
Contain 2 ORFs
ORF1, similar to gag
ORF2, similar to pol

In humans, LINEs account for

20% of genome !
 DNA methylation and
trinucleotide repeat expansion
Species specific methylation of A and C residues in DNA
to: N6-methyladenine (m6A)
N4-methylcytosine (m4C)
5-methylcytosine (m5C)
 DNA methylation

Bacterial DNA is methylated at own restriction site

E.coli, Dam methyltransferase (dam MTase), A in GATC

Dcm MTase bith C in CCA/TGG at pos 5
both palindromic, mismatch repair and oriC

Methyl groups project into major groove of B-DNA,

interact with DNA-binding proteins
 The MTase reaction occurs via a covalent
intermediate in which the target base is flipped out
Methylation uses SAM, S-adenosylmethionine as methyl
donor via a Cys thiolate attack, uses base flipping
Inhibited by 5-fluorocytosine
Base flipping
 DNA methylation in eukaryotes
functions in gene regulation
5-methylcytosine is the only methylated base in most
eukaryotes
Modification in largely in GC dinucleotide
CG is present at 1/5 of statistical expectation
Upstream regions of many genes have CpG island
DNA methylation in eukaryotes

Experimental assessment:
Comparative southern blot of DANN cut with
HpaII, cleaves CCGG, but not C-m5C-GG and
MspI, cleaves both

Identification of m5C residues through bisulfite

sequencing
DNA is reacted with bisulfite (HSO3-) which
deaminates C to U, but not m5C,
followed by PCR amplification:
copies U to T and m5C to C
Sequence and compare to untreated
DNA methylation in eukaryotes (2)
Methylation switches off eukaryotic gene expression,
particularly when methylation occurs in promoter region
For example, globin genes are less methylated in
erythroid cells

Recognized by methyl-CpG binding domain (MBD)

May also affect chromatin packaging
 DNA methylation in
eukaryotes is self-
perpetuating
Maintenance of methylation after
replication -> inherited,

Epigenetic inheritance:
Non-Mendelian inherited information

By DNMT1, which has preference

for hemimethylated sites
DNMT1 null mice die early in embr.
devel.
 Methylation is dynamic
Pattern of DNA methylation varies in early embryological
development:

Methylation levels high in gamets (sperm, ova) but

nearly eliminated in blastocyst stage
Methylation then rises again till gastrula stage
when it reaches that found in adults, remain constant
Except germ line cells, remain unmethylated

Pattern of expression differs in embryonic and somatic

cells
=> Explains high failure of cloning experiments, few
survivers, early death, abnormalities, large size
 Genomic imprinting results from
differential DNA methylation
Difference in maternal and paternal inheritance:
Mare x Male donkey -> mule
Female donkey x stallion -> hinny
Both are sterile

mule hinny
Maternal and paternal genes are differentially expressed
= genomic imprinting, only in mammals
No embry from transplant of two male or female pronuclei
 DNA methylation is associated
with cancer

Most prevalent mutation is is m5C to T, covert

proto-oncogens to oncogens or inactivate tumor
suppressors
 Several neurological diseases are
associated with trinucleotide repeat
expansion
Fragile X syndrome: mental retardation, long narrow face
1 in 4500 males, 1 in 9000 females
Activated by passage through female
Affects FMR1 gene, which contains (CGG)n, n=6-60 in
5’ region, n can increase from 60 to 200 = premutation
Can the expand upon transmission to a daughter to >200
= full mutation

Expansion arises through slippage during replication

FMR1 is unmethylated in normal individuals

But is methylated when premutation is maternally
transmitted
 Other important trinucleotide
repeat diseases
Huntington’s disease (HD), 1 in 10’000, onset at age
of approx. 40, 18-year course, fatal
Protein huntingtin contains (CAG)n repeats (Gln)
Normal 11-34, sick 37-86
Repeat length is unstable, changes in >80% meiotic
transmissions
Number of repeats inversely correlates with age of onset
polyGln aggregates as β sheets
Neurons contain inclusions
The loop-out mechanism for the alteration
of the number of consecutive triplet
repeats in DNA through its replication