DNA replication
Replication represents the duplication of the genetic information encoded in DNA that is
the crucial step in the reproduction of living
organisms and the growth of multicellular
organisms. Replication is semiconservative
so that each new DNA double helix consists
of one parental template strand hydrogen
bonded along its entire length by basepairing to a newly synthesized strand (Fig.
1).
Fig. 1. Model of replication
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Components required for replication:
- DNA as template;
- deoxyribonucleoside
triphosphates
(dNTP) for synthesis of DNA;
- ribonucleoside triphosphates (NTP)
for synthesis of primers;
- proteins for unwinding the doublehelix of DNA;
proteins for initiation of replication;
proteins for polymerization of nucleotides.
In the process of replication participate a lot of proteins, including enzymes:
- DNA helicases - enzymes that unwind DNA to facilitate separation of the two strands of
the duplex.
- Primase – a RNA-polymerase that synthesizes the short RNA primers for DNA
replication using DNA as a template.
- Topoisomerases – enzymes that catalyse the interconversion of different topological
isomers of DNA that involves the transient breakage of one (type I) or both strands (type
II) of DNA and can result in the removal of negative or positive supercoils from DNA or
the introduction of negative supercoils.
- DNA-polymerazes – enzymes that synthesis new strands of DNA in the direction 5’-3’
on the bases of templates. All polymerases can only continue strands, and for initiation
of activity need free 3’ ends. Some DNA-polymerases have also exonuclease activity:
can remove nucleotides from the end in the direction 5’-3’ or 3’-5’.
- DNA-ligases – enzymes that bind the fragments of DNA with 3’-5’ phosphodiester
bonds.
- SSB protein – bind single-stranded DNA regions.
Replication initiates at specific replication origins (ORI), to generate two replication
forks, which are elongated bidirectionally by multienzyme replication complexes - the
replisome. Prokaryotic circular DNAs are replicated from a single origin, but eukaryotic DNAs
have multiple origins of replication so that the large chromosomes can be replicated sufficiently
rapidly. A region or unit of a chromosome served by a single origin of replication is called
replicon.
Mechanisms of replication
The two strands of a DNA double helix are antiparallel, that is, they run in opposite
directions so that the terminal 5'PO4 of one strand is opposite the terminal 3'OH of the other.
However, DNA polymerases can only add nucleotides to the 3'OH group of a polynucleotide
chain. These constraints are overcome by the use of RNA primers and a semi-discontinuous
Replication
mode of synthesis. With rare exceptions, DNA chains are initiated by synthesis of short RNA
primers, which are initiated de novo by RNA polymerases known as primases using the DNA
strand as a template. These provide a 3' hydroxyl group from which DNA polymerases can
synthesize new DNA. Since the strands are antiparallel and DNA polymerases can only add to
the 3' end, synthesis of both strands at a single replication fork requires two mechanisms. One
strand, the leading strand, is synthesized continuously by the repeated addition of nucleotides to
its 3' end, whereas the other lagging strand is synthesized discontinuously in segments called
Okazaki fragments which are about 1000 nucleotides long in bacteria or 150 nucleotides in
eukaryotes. Gaps between the fragments are subsequently filled to form the second continuous
DNA strand (Fig. 2).
Mechanism of replication
DNA polymerases
DNA polymerases polymerize deoxyribonucleoside triphosphates into DNA by a
condensation reaction that forms a phosphodiester bond linking the 3-OH of the sugar
component of one nucleotide and the 5-OH of the sugar of the next with the release of
pyrophosphate. There are several types of DNA polymerase in any organism. In Escherichia
coli, DNA polymerase III synthesizes both leading and lagging strands. The gaps between
Okazaki fragments are filled by DNA polymerase I.
In eukaryotes, chromosomal DNA is replicated by three DNA polymerases α, δ, and ε.
Polymerase α contains an integral primase. Polymerase α is required for lagging strand synthesis
and possibly for the initial priming of leading strands too. Polymerase δ is required for leading
strand synthesis. Mitochondrial DNA is synthesized by polymerase γ. Most DNA polymerases,
but probably not polymerase α, contain a proofreading exonuclease which excises
misincorporated bases, increasing the fidelity of template copying.
Initiation of replication
Prokaryotes initiate DNA replication at unique sites, called origins of replication. In
eukaryotes multiple origins are used, so that eukaryotic chromosomes are replicated by many
replication forks simultaneously.
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Replication
In prokaryotes and some animal viruses, and presumably elsewhere, replication origins
are recognized by sequence-specific binding proteins,
which can locally unwind a specific region of the
double helix allowing replication to initiate.
Initiation occurs in some steps:
- recognition of ORI by special proteins;
- DNA-helicase unwinds DNA;
- Primase synthesis a short fragment of RNA –
primer;
- DNA-polymerase adds new nucleotides at 3’
end (Fig. 3).
All of proteins which participate at initiation
form the promosome.
Unidirectional replication occurs in some
phages and some prokaryotic circular DNAs replicate
by a rolling circle mechanism (Fig. 4). Fidelity of
DNA replication is ensured by proofreading and DNA
repair mechanisms. Regulation of DNA replication is a
critical control point in cell proliferation.
Fig. 3. Initiation of replication
Fig. 4. Rolling circle mechanism of replication
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Replication
Termination and telomeres
When two converging replication forks meet, their nascent strands are joined. DNA
topoisomerase II is required to unwind the two progeny DNA molecules from around each other
in these final stages. The ends of
the long linear chromosomes of
eukaryotes, called telomeres, are
replicated by a different
mechanism. They consist of
many copies of a short repeating
sequence which are added by
the enzyme telomerase. This
enzyme contains an RNA
template which it copies into
DNA
to
complete
the
chromosome ends (Fig. 5).
Mitochondrial DNA has
two ORI – one for light strand
(L) and the other for heavy
starnds (H).
Fig. 5. Replication of telomeres
DNA repair
DNA repair represents a range of cellular responses associated with restoration of
primary structure of DNA.
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Mechanisms of alteration of DNA molecules:
base substitution during replication
base changes resulting from chemical instability of bases
alterations resulting from the action of other chemical and environmental agents.
The possible defects in DNA molecules:
An incorrect base in one strand cannot form hydrogen bond with the corresponding base
in the other.
▪ Defect can result from replication
errors or
▪ Deamination of C to U, followed by
replacing of U by T in subsequent
rounds of replication.
- Missing bases – depurination
▪ Alkylating agents (used in cancer
treatment) react with G and weak Nglycosidic bond.
- Altered bases
▪ Chemical and physical agents can break
purine and pyrimidine rings. Formation
of thymine dimmers (Fig. 6).
- Single-strand breaks.
▪ Peroxides, Fe2+, Ca2+,
Ionizing
radiation can attack the phosphodiester
Fig. 6. Formation of pyrimidinic dimers
bonds
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Replication
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▪ DNases present in cells make phosphodiester scissions.
Double-strand breaks.
▪ Highly ionizing radiations can produce numerous single strand breaks and as
result – double-strand breaks.
Mechanisms of DNA repair
Excision repair – excision of damaged bases
Recombination repair – reconstruction of DNA from undamaged fragments
SOS repair – in prokaryotic cells. Enzymes synthesis DNA without fidelity of
replication.
Types of reparation
Photoreactivation - direct repair, when enzymatic cleavage of thymine dimers is activated by
visible light. Photolyase was discovered in different species, but is active only in bacteria.
A
B
Fig. 7. A - Base excision repair (BER); B - Nucleotide excision repair (NER)
Repair of alkylation damages by MGMT – O6-methyl-guanine DNA methyltransferase (in
human). 20% of human tumor cell have reduced MGMT activity.
Base excision repair (BER) – reparation of single nucleotide damage (Fig. 7.A).
▪ DNA-glycosylase removes the damaged (modified, fragmented) base from DNA
▪ AP-lyase makes an excision at 3’ end (AP – apurinic/apyrimidinic sites)
▪ AP-hydrolase incises at 5’ end
▪ DNA-polymarease fills in the gap beginning with 3’ end
▪ DNA-ligase seals the nick.
Nucleotide excision repair (NER) – reparation of bulky DNA damages (Fig. 7.B)
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Replication
▪
▪
▪
▪
A complex of proteins (XPA-PRA) recognizes the damaged fragment
Excinuclease cuts the 5th phosphodiester bond 3’ and 24th phosphodiester bond
5’ to the lesion (single-strand incisions)
The gap is filled by DNA-polymerases δ and ε
DNA-ligase seals the nick.
Mismatch repair (MMR) reparation of misincorporations during replication and the
mismatches resulted of deamination of 5-methylcytosine to uracil
▪ An unknown enzyme excises the mismatch bases
▪ The alkylating agents induced expression of DNA-polymerase β which fills in the
gap
▪ DNA-ligase seals the nick.
Recombination repair (Fig. 8)
- Excision of damaged fragment of DNA
- Transport of homologous fragment from another molecule
- Legation of the fragments.
Fig. 8. Recombination repair
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