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DNA replication
Semi-conservative mechanism
1958, Meselson & Stahl
15N
labeling experiment
Rosalind Franklin (1920-1958)
Maurice Wilkins (1916-2004)
Francis Crick (1916-2004)
James Watson (1928-)
Discovery of DNA structure
1962 Nobel Prize
The substrates of DNA synthesis
dNTPs – dATP, dGTP, dCTP, dTTP
Direction: 5’-3’
5’
3’
T CA
5’PPP
C
OH
+
ppp
OH
T CA C
5’PPP
OH 3’
+
ppi
3’
5’ ???
G
ppp
A T
OH
+
G A
5’ ppp
C G
5’ ppp
T
OH 3’
C G
OH 3’
G
ppp
A T
OH
+
5’ ppp
5’ ppp
A
G
ppp
???
C G
T
OH 3’
C G
OH 3’
Proofreading???
ppp
A T C G
T C G
A
+ p
ppp
OH
OH
pp
T C G
p
T C G
OH
pp p
OH
Replicon is any piece of DNA which replicates
as a single unit. It contains an origin and
sometimes a terminus
Origin is the DNA sequence where a replicon
initiates its replication.
Terminus is the DNA sequence where a replicon
usually stops its replication
All prokaryotic chromosomes and many bacteriophage
and viral DNA molecules are circular and comprise
single replicons.
There is a single termination site roughly 180o opposite
the unique origin.
The long, linear DNA molecules of eukaryotic
chromosomes consist of mutiple regions, each with
its own orgin.
A typical mammalian cell has 50000-100000 replicons
with a size range of 40-200 kb. When replication
forks from adjacent replication bubbles meet, they
fuse to form the completely replicated DNA. No
distinct termini are required
Semi-discontinuous replication
Experimental evidences
[3H] thymidine pulse-chase labeling experiment
1. Grow E. coli
2. Add [3H] thymidine in the medium for a few second, spin down
and break the cell to stop labeling, analyze and find a large fraction
of nascent DNA (1000-2000 nt) = Okazaki fragments
3. Grow the cell in regular medium then analyze, the small fragments
join into high molecular weight DNA = Ligation of the Okazaki
fragments
Back
Bacterial DNA replication
Experimental systems
1. Purified DNA: smaller and simpler
bacteriophage and plasmid DNA molecules
(ΦX174, 5 Kb)
2. All the proteins and other factors for its
complete replications
Initiation: oriC
Study system
the E. coli origin locus oriC is cloned into
plasmids to produce more easily studied
minichromosomes which behave like
E.coli chromosome.
1. oriC contains four 9 bp binding sites for the initiator
protein DnaA. Synthesis of DnaA is coupled to growth rate
so that initiation of replication is also coupled to growth
rate.
2. DnaA forms a complex of 30-40 molecules, facilitating
melting of three 13 bp AT-rich repeat sequence for DnaB
binding.
3. DnaB is a helicase that use the energy of DNA hydrolysis
to further melt the double-stranded DNA .
4. Ssb (single-stranded binding protein) coats the unwinded
DNA.
5. DNA primase attaches to the DNA and synthesizes a short
RNA primer for synthesis of the leading strand.
6. Primosome DnaB helicase and DNA primase
Unwinding
Positive supercoiling: caused by removal of
helical turns at the replication fork.
Resolved by a type II topoisomerase called
DNA gyrase
Elongation
DNA polymerase III holoenzyme
1. A dimer complex, one half synthesizing the leading strand
and the other lagging strand.
2. Having two polymerases in a single complex ensures that
both strands are synthesized at the same rate
3. Both polymerases contain an
α-subunit---polymerase
ε-subunit---3’
5’ proofreading exonuclease
β-subunit---clamp the polymerase to DNA
other subunits are different.
Replisome
in vivo DNA polymerase holoenzyme dimer, primosome
(helicase) are physically associated in a large complex to
synthesize DNA at a rate of 900 bp/sec.
Other two enzymes during Elongation
1. Removal of RNA primer, and gap filling with DNA pol I
2. Ligation of Okazaki fragments are linked by DNA ligase.
Prokaryotic DNA replication
Termination and segregation
Terminus
containing several terminator sites (ter) approximately
180o opposite oriC.
Tus protein
ter binding protein, an inhibitor of the DnaB helicase
Topoisomerase IV
a type II DNA topoisomerase, function to unlink the
interlinked daughter genomes.
Eukaryotic DNA replication
Experimental systems
1. Small animal viruses (simian virus 40, 5 kb) are good
mammalian models for elongation (replication fork) but
not for initiation.
2. Yeast (Saccharomyces cerevisiae): 14 Mb in 16
chromosomes, 400 replicons, much simpler than
mammalian system and can serve as a model system
3. Cell-free extract prepared from Xenopus (frog) eggs
containing high concentration of replication proteins and
can support in vitro replication.
Cell cycle
Entry into the S-phase
Cyclins
CDKs (Cyclin-dependent protein kinases)
DNA Replication
DNA replication is semi-conservative, one strand serves as the template for the second strand.
Furthermore, DNA replication only occurs at a specific step in the cell cycle.
The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle.
Stage
G1
S
G2
M
Activity
Growth and increase in cell size
DNA synthesis
Post-DNA synthesis
Mitosis
Duration
10 hr
8 hr
5 hr
1 hr
DNA replication has two requirements that must be met:
1.
2.
DNA template
Free 3' -OH group
Origin and initiation
1. Clusters of about 20-50 replicons initiate simultaneously at
defined times throughout S-phase
Early S-phase: euchromatin replication
Late S-phase: heterochromatin replication
Centromeric and telomeric DNA replicate last
2. Only initiate once per cell cycle
Licensing factor
required for initiation
inactivated after use
can only enter into nucleus when the nuclear envelope dissolves at mitosis
Electron Microscopy of replicating DNA reveals
replicating bubbles.
3. Individual yeast replication origins (ARS) have been
cloned into prokaryotic plasmids which allow these
plasmids to replicate in yeast (an eukaryote).
ARSs autonomously replicating sequences
Minimal sequence 11 bp
[A/T]TTTAT[A/G]TTT[A/T] (TATA box)
4. ORC (origin recognition complex) binds to ARS, upon
activation by CDKs, ORC will open the DNA for
replication.
Elongation
1. Replication fork
- unwinding DNA from nucleosomes: 50 bp/sec
- need helicases and replication protein A (RP-A)
- new nucleosomes are assembled to DNA from a mixture
of old and newly synthesized histones after the fork passes
2. Elongation
Three different DNA polymerases are involved
1) DNA pol α contains primase activity and synthesizes RNA
primers for the leading strands and each lagging strand
fragments. Continues elongation with DNA but is replaced
by the other two polymerases quickly.
2) DNA pol δ on the leading strand that replaces DNA pol α.,
can synthesize long DNA
3) DNA pol ε on the lagging strand that replaces DNA pol α.,
synthesized Okazaki fragments are very short (135 bp in
SV40), reflecting the amount of DNA unwound from each
nucleosome.
Nuclear matrix
1. A scaffold of insoluble protein fibers which acts as an
organizational framework for nuclear processing, including
DNA replication, transcription
2. Replication factories
containing all the replication
enzymes and DNA associated
with the replication forks
in replication
BudR labeling of DNA
Telomere replication
Telomerase
1. Contains a short RNA molecule as telomeric DNA
synthesis template
2. Telomerase activity is repressed in the somatic cells of
multicellular organism, resulting in a gradual shortening of
the chromosomes with each cell generation, and ultimately
cell death (related to cell aging)
3. The unlimited proliferative capacity of many cancer cells is
associated with high telomerase activity.
Telomerase activity is repressed in somatic cells
of multicelluar organisms resulting in a gradual
shortening of the chromosome with each cell
generation. As this shortening reaches
informational DNA, the cells senesce and die.
When telomerase
activity is repressed
informational DNA
cell division
cell dies or does not divide
Mutagenesis
Mutation
Permanent, heritable alterations in the base sequence of DNA
Reasons
1. Spontaneous errors in DNA replication or meiotic recombination
2. A consequence of the damaging effects of physical or chemical
mutagens on DNA
Point mutation
A singe base change: transition, transversion
The effects of point mutation
Phenotypic effects
Noncoding DNA
Nonregulatory DNA
3rd position of a codon
Silent mutation
Coding DNA
altered AA
Missense mutation
Yes or No
Coding DNA stop codon
Truncated protein
Nonsense mutation
Yes
No
Insertions & deletions
The addition or loss of one or more bases in a DNA region
Frameshift mutations
The ORF of a protein encoded gene is changed so that the
C-terminal side of the mutation is completely changed.
Genetic polymorphisms
Caused by accumulation of many silent and other
nonlethal mutations
Replication fidelity
Important for preserve the genetic information from one
generation to the next, spontaneous errors in DNA replication
is very rare, e.g. one error per 1010 base in E. coli.
Molecular mechanisms for the replication fidelity
1.
2.
3.
4.
DNA polymerase: Waston-Crick base pairing
3’ 5’proofreading exonuclease.
RNA priming: proofreading the 5’end of the lagging strand
Mismatch repair
Mutagens
Causing DNA damage that can be converted to mutations.
Physical mutagens
High-energy ionizing radiation
X-rays and γ-rays
strand breaks and base/sugar destruction
Nonionizing radiation
UV light
pyrimidine dimers
Chemical mutagens
Base analogs
direct mutagenesis
Nitrous acid
deaminates C to produce U
Alkylating agents
Arylating agents
indirect-lesion mutagenesis
Intercalators: e.g. EB
Mutagenesis
The molecular process in which the mutation is
generated.
Note the great majority of lesions introduced by chemical and physical mutagens
are repaired by one or more of the error-free DNA repair mechanisms before
the lesions is encounter by a replication fork
Direct mutagenesis
The stable, unrepaired base with altered base pairing
properties in the DNA is fixed to a mutation during DNA
replication.
Indirect mutagenesis
The mutation is introduced as a result of an error-prone repair.
Translesion DNA synthesis
to maintain the DNA integrity but not the sequence accuracy
when damage occurs immediately ahead of an advancing
fork, which is unsuitable for recombination repair, the
daughter strand is synthesized regardless of the the base
identity of the damaged sites of the parental DNA.
DNA damage and repair
Oxidative
damage
1. Occurs under
normal condition
2. Increased by
ionizing radiation
physical mutagens
DNA lesions
Alkylation
Alkylating agents
Chemical mutagens
Bulky adducts
UV light
physical mutagens
Carcinogen
Chemical mutagens
Biological effects of the unrepaired DNA lesions
Physical distortion of
the local DNA structure
Altered chemistry of the
bases
Blocks replication
and/or transcription
Allowed to Remain in the
DNA
Lethal
A mutation could become
fixed by direct or indirect
mutagenesis
Mutagenic
Spontaneous DNA lesions
1. Inherent chemical reactivity of the DNA
2. The presence of normal, reactive chemical species within
the cell
- Deamination
C
U
methylcytosine
T
- Depurination
break of the glycosylic bond, non-coding lesion
- Depyrimidine
Oxidative damage
1. occurs under NORMAL conditions in all aerobic cells due
to the presence of reactive oxygen species (ROS), such as
superoxide, hydrogen peroxide, and the hydroxyl radicals
(•OH).
2. The level of this damage can be INCREEASED by
hydroxyl radicals from the radiolysis of H2O caused by
ionizing radiation
Alkylation
1. Electrophilic chemicals adds alkyl groups to various
positions on nucleic acids
2. Distinct from those methylated by normal methylating
enzymes.
3. Typical alkylating agents:
MMS methylmethane sulfonate
EMS ethylmethane sulfonate
ENU ethylnitrosourea
Bulky adducts
1.
DNA lesions that distort the double helix and
cause localized denaturation, for example
pyrimidine dimers
arylating agents adducts
2. These lesions disrupt the normal function of the
DNA
DNA repair
Photoreactivation
1. Monomerization of cyclobutane pyrimidine dimers by
DNA photolyases in the presence of visible light
2. Direct reversal of a lesion and is error-free
Alkyltransferase
1. Removing the alkyl group from mutagenic O6-alkylguanine
which can base-pair with T. The alkyl group is transferred to the
protein itself and inactivate it.
2. Direct reversal of a lesion and is error-free
3. In E.coli, The response is adaptive because it is induced by low
levels of alkylating agents and gives increased protection
against the lethal and mutagenic effects of the high doses
Excision repair
1. Including
nucleotide excision repair (NER)
base excision repair (BER)
2. Ubiquitous mechanism repairing a variety of lesions.
3. Error-free repair
Nucleotide excision repair (NER)
1. An endonuclease
cleaves DNA a precise
number of bases on
both sides of the lesions
(e.g. in E.coli, UvrABC
Endonulcease removes
pyrimidine dimers)
2. Excised lesion-DNA
fragment is removed
3. The gap is filled by
DNA polymerase I
and sealed by ligase
Base excision repair
(BER)
Mismatch repair
A specialized form of excision repair which
deals with any base mispairs produced
during replication and which have escaped
proofreading
The parental strand is methylated at N6 position of all
As in GATC sites, but methylation of the daughter
strand lag a few minutes after replication
MutH/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
Essay questions
1.
How to explain the mechanisms of semi-conservative
replication and semi-discontinuous replication? How
to verify them by experiments?
2.
How about the differences between prokaryotic and
eukaryotic DNA replication?
3.
How about the main types of DNA damage? and the
main repair mechanisms?
DNA recombination
- Homologous recombination
- Site-specific recombination
- Transposition
An important reason for variable DNA sequences
among different populations of the same species
Homologous recombination
- The exchange of homologous regions between two
DNA molecules
In diploid eukaryotes, it commonly occurs during meiosis
1. Homologous duplicated chromosomes line up in
parallel in metaphase I.
2. The nonsister chromatids exchange equivalent sections
by crossing over.
Crossing over
Haploid prokaryotes recombination
Occurs between the two homologous duplex
-
between the replicated portions of a partially
duplicated DNA
- between the chromosomal DNA and acquired
“foreign” DNA, like plasmids or phages
Nick formation
RecA-ssDNA
filament
Recombination-based DNA repair
Site-specific recombination
1. Exchange of non-homologous but specific pieces
of DNA
2. Mediated by proteins that recognize specific DNA
sequences.
Bacteriophage λ insertion
1. λ -encoded integrase (Int): makes staggered cuts in
the specific sites
2. Int and IHF (integration host factor encoded by
bacteria): recombination and insertion
3. λ -encoded excisionase (XIS): excision of the
phage DNA
Antibody diversity
H and L are all encoded by three gene segments: V, D, J
V
D
J
Two heavy chains (L)
250
15
5
Two light chains (H)
250
4
Enormous number (>108) of different H
and L gene sequences can be produced by
such a recombination
Transposition
1. Requires no homology between sequences nor
site- specific
2. Relatively inefficient
3. Require Transposase encoded by the transposon
Transposons
E. coli
- IS elements/insertion sequence
1-2 kb, comprise a transposase gene flanked by a short inverted
terminal repeats
- Tn transposon series
carry transposition elements and β-lactamase
(penicillin resistance)
Eukaryotic transposons
many are retrotransposons
Yeast Ty element encodes protein similar to RT
(reverse transcriptase)