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The chemical reaction catalyzed by DNA polymerase I.
In a reaction discovered by Kornberg (Nobel 1959) et al.
a single nucleotide is added to the growing complement of the DNA template,
using a nucleoside triphosphate as the substrate.
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The active form of DNA polymerase III is called a holoenzyme,
a dimer with 10 different polypeptide subunits.
The α (alpha), ε (epsilon) and θ (theta) subunits make up the core enzyme to perform polymerization and proofreading.
The γ (gamma) complex is involved in "loading" the enzyme onto the template at the replication fork.
The β (beta) subunit serves as a "clamp" and prevents the core enzyme from falling off the template during polymerization.
The τ (tau) subunit functions to dimerize two core polymerases facilitating simultaneous synthesis of both strands of the helix.
The holoenzyme and several other proteins at the replication fork together form a huge complex
called the replisome.
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Energy for the reaction is driven by the exergonic (energy-releasing)
hydrolysis
of the dNTP, releasing inorganic pyrophosphate.
video
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These DNA polymerases cannot initiate DNA synthesis,
but can elongate an existing DNA or RNA strand (primer).
Polymerase I fills gaps in the synthesized strand and also removes the primer
by its 5' to 3' exonuclease activity.
Polymerase III
is the main enzyme for 5' to 3' polymerization in vivo.
All three possess 3' to 5' exonuclease activity which allows proofreading.
Polymerases II, IV and V are involved in DNA repair.
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Since polymerization by DNA polymerase III occurs only in the 5' to 3' direction,
elongation along the two antiparallel strands are dissimilar.
Synthesis along the leading strand of a replication fork can occur continuously, while
synthesis along the lagging strand must be discontinuous, occurring in Okazaki fragments,
each with an RNA primer.
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Summary of DNA synthesis in bacteria.
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Okazaki fragments on the lagging strand.
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Transmission electron micrograph of human DNA from a HeLa cell,
illustrating replication forks and the associated replication bubble.
HeLa cells were derived from cervical cancer cells taken from
"Helen Lane", who died from her cancer in 1951, but her cells,
which possess high telomerase activity, have continued to divide in culture.
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Initiation of DNA synthesis begins when primase builds a short RNA primer
in the 5' to 3' direction that is complementary to the template strand of the helix.
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b
c
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Bidirectional replication of the E. coli chromosome
starts at a fixed origin of replication (oriC).
As the DNA unwinds, two replication forks (arrows) move away from the origin, forming a replication bubble.
The forks eventually merge as DNA replication is completed at a termination region (ter).
video
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The precursor dNTP contains three phosphate groups attached to the 5'-carbon of deoxyribose.
As the two terminal phosphates are cleaved, the remaining phosphate is linked to the
3'-OH group of the growing chain.
Thus, chain elongation occurs in the 5' to 3'
direction by adding one nucleotide at a time to the 3' (-OH)
end.
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DNA is synthesized by unwinding the helix, then using base-pairing rules
to replicate each strand.
The process is semiconservative: each replicated double helix consists of one "old" and one "new" strand.
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Polymerization occurs concurrently on both strands
by a single DNA polymerase III holoenzyme.
The lagging template strand is looped at the replication fork,
allowing each core enzyme of the dimer to add bases in the 5' to 3'
direction.
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Three eukaryotic DNA polymerases catalyze reactions in DNA replication, while others are involved in repair.
Pol α (alpha) synthesizes the RNA primers during initiation. Then, in a process called polymerase switching, it is replaced by Pol δ (delta), which performs the main task of concurrent elongation of both strands.
Pol ε (epsilon) is the other enzyme involved in nuclear DNA synthesis, possibly
playing a role in binding to the origin or synthesis of the lagging strand.
Pol γ (gamma) is encoded by a nuclear gene though its function is synthesis of
mitochondrial DNA.
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A base-pair mismatch occurs in one of the two homologs during heteroduplex formation in meiosis.
During excision repair, one of the two mismatches is removed and the complement is synthesized,
leading to possible gene conversion.
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A model for homologous recombination.
RecA protein in E. coli
may be an enzyme that promotes such exchange of reciprocal single-stranded DNA molecules.
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genetic_recombination3.html: 11_18-genetic_recombination3.jpg
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Unwinding of the bacterial helix begins when
monomers of the protein DnaA bind to DNA sites containing repeating sequences of
9 and 13 bases (called 9mers and 13mers).
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DnaB and DnaC helicase proteins open the helix by breaking
hydrogen bonds between the bases, denaturing the double helix and forming a replication bubble.
Single-stranded binding proteins (SSBPs) stabilize the unwound helix, preventing renaturing of the helix.
The double helix becomes supercoiled ahead of the replication fork. This supercoiling is relaxed by DNA gyrase.
Energy to break the hydrogen bonds is provided by the hydrolysis of
ATP.
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Semiconservative synthesis of the leading strand in a linear chromosome
can proceeds normally to the end of the double helix.
However, after the last RNA primer is removed from the lagging strand, there is no fragment to provide the free 3'-OH for DNA polymerase to elongate.
A gap remains on the lagging strand, leading to shortening of the chromosome during each round of synthesis.
This chromosome shortening may play a role cellular aging of somatic cells,
and must be avoided in germ cells.
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Eukaryotic chromosomes are associated with proteins called histones, forming
complexes of nucleosomes.
These nucleosomes have to be opened up to initiate DNA synthesis.
The histones also need to be duplicated, and then reassociated with DNA into nucleosomes
during the S phase of the cell cycle.
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Eukaryotic chromosomes contain multiple replication origins that form multiple replication bubbles.
This allows the larger genomes of eukaryotic cells to be replicated in multiple replicons
and completed in hours.
For example, Drosophila has 40-100 times as much DNA as E. coli.
The origins reside within an AT-rich regions, where a helicase enzyme unwinds the double helix.
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| =To replicate a double-stranded DNA molecule, three modes are possible. | ||||
|---|---|---|---|---|
|
Conservative: the original helix is thus "conserved" after synthesis. |
Semiconservative: each replicated double helix consists of one "old" and one "new" strand. |
Dispersive: segments of the parental strands are dispersed into the new strands. |
||
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The β (beta) subunit also forms a dimer that
serves as a "clamp" to keep the core enzyme bound to the DNA
tempplates.
Thus the entire holoenzyme moves along the parent duplex as a sliding clamp,
advancing the replication fork.
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The enzyme telomerase is capable of synthesizing short repeating sequences of DNA,
called telomeres, at the 3' end of an eukaryotic chromosome, preventing chromosome shortening,
especially in germ cells.
This enzyme is a ribonucleoprotein with RNA segments that serve as template for the reverse transcription of the DNA sequences.
These repeats fold back on themselves by forming unorthodox G-G hydrogen bonds.
The gap is filled by a DNA polymerase and ligase.
The hairpin loop is then be cleaved off, preserving the original duplex.
This allows gametes and malignant cells, as well as some "immortal" cultured cells, to continue duplicating the linear DNA.