Chapter 30:
DNA Replication, Repair,
and Recombination
1.
2.
3.
4.
5.
6.
DNA Replication: An overview
Enzymes of Replication
Prokaryotic Replication
Eukaryotic Replication
Repair of DNA
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
Discontinous !
Okazaki fragments:
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 Nterm.
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.
b.
c.
d.
e.
Specific cut at + strand
Extension of + strand
Tandem-linked + strands
Separation by endonuclease
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 Dloops
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 FADHElectron 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 hemiMethylated 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.
2.
3.
4.
ssDNA nick
Strand invasion
Branch migration
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 RecAmediated 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 requiring 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 singlestrand 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 intermediate
1. Resection of DS ends
2. DNA dynthesis and ligation
3. Resolution of 2 Hol.j.
Transposition and sitespecific 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. antibiotic 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.
2.
3.
4.
5.
Transposase binding
Dimerization
Synaptic complex
Target capture
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 sitespecific
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 sitespecific 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 selfperpetuating
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