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Takusagawa’s Note
Chapter 24 (Summary)
Summary of Chapter 24
1. DNA is replicated in a semiconservative fashion.
• By using heavy DNA containing 14N and ultra-centrifugation, the semiconservative
replication has been proved.
• New strand is synthesized from 5’→ 3’ direction. The template is read from 3’→5’
direction.
• The reaction is catalyzed by DNA polymerase, and the energy is from the substrate
hydrolysis.
• Substrates are tri-deoxynucleotides (dATP, dGTP, dCTP, dTTP), and PPi are released.
2. DNA replication of E. coli
• A circular DNA initially shows a replication eye, and then θ structure.
• In general, replication is bi-directional and thus has two growth forks.
• Leading strand --- Synthesis on the 3’→5’ template.
• Lagging strand --- Synthesis on the 5’→3’ template.
• Leading strand is continuously synthesized, whereas lagging strand is discontinuously
synthesized.
• Discontinuously synthesized DNA fragments are called “Okazaki fragments”.
• Each Okazaki fragment contains a small amount of RNA --- indicates that RNA is a primer in
replication.
• RNA primers are removed, the gaps are filled with DNA, and the fragments are ligated.
3. DNA polymerase I (E. coli)
• DNA polymerase I (Pol I) is a repair enzyme, and has
• 5’ → 3’ polymerizing activity.
• 3’ → 5’ exonuclease activity. 3’→5’ exonuclease cleaves the 3’-end residue of DNA.
• 5’ → 3’ exonuclease activity.
• Pol I has an editing function --- A nucleotide that is erroneously incorporated is removed by
3’→5’ exonuclease function.
• Pol I can remove the primer at the 5’-end and the damaged section by 5’→3’ exonuclease
function.
• Pol I is composed of two domains, N-ter “small fragment” which has 5’→3’ exonuclease
function and C-ter “large fragment” which has polymerase and 3’→5’ exonuclease function.
• The large fragment is called Klenow fragment. It looks like a right hand shape.
• The major roles of Pol I are to repair the damaged sections and to replace the primer RNA
from DNA.
4. Prokaryotic DNA polymerase III (Pol III)
• The holoenzyme consists of at least 10 subunits, (αεθ)3β2.
• The two β-subunits form a ring structure, and a double stranded DNA passes through the
ring.
• Without β-subunits, Pol III core synthesizes only 10~15 residues.
• Pol III is the major DNA replicator.
• The other important enzymes for E. coli replication are:
1. Ligase --- joins Okazaki fragments.
2. Topoisomerase --- relaxes positive supercoil and/or generates negative supercoil.
3. Primase --- make primer RNA
4. SSB (single strand binding protein) --- keep strands apart.
5. DnaA --- recognizes the negatively supercoiled oriC and makes a complex with oriC.
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Takusagawa’s Note
Chapter 24 (Summary)
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6. DnaB (helicase) --- unwinding DNA helix.
7. DnaC --- help DnaB to bind to DNA.
Initiation of replication
1. DnaA proteins activated by ATP bind on the 9-bp sections of the oriC.
2. The oriC section wraps the DnaA proteins by forming a negative supercoil.
3. P1 insensitive segment (13-bp) located near oriC melts to two single strands.
4. DnaB bind to the single stranded DNA with aids of DnaC and ATP energy.
5. Primase and RNA polymerase bind to the single strand DNA, and attach a primer RNA.
6. Two Pol III connected to each other bind on each single strand DNA, and synthesize DNA
at the same direction.
The initiation is controlled by:
• Level of DnaA.
• Adenine methylation (N6-methyl A) --- Methylation is required for good initiation.
Termination:
• Two sets of three termination sites are faced each other flanked by ~230 bp.
• The first sites (TerA and TerC) are the major termination sites, whereas the TerD, TerF and
TerB, TerF are backup termination sites.
• Tus proteins from tus gene bind the termination sites and prevent the DnaB (helicase)
action.
The catenated DNA strands are unlinked by topoisomerases.
High fidelity of replication arises from five sources:
1. Balanced level of dNTPs.
2. Pol III has high base recognition by base-pairing and shape recognition.
3. Pol III has editing function (3’→5’ exonuclease function).
4. Cells contain repair mechanism --- Pol I.
5. Use of RNA primer --- Most errors occur at the initiation stage, but the RNA primers are
removed.
Why both DNA strands are synthesized 5’→3’ direction?
Because the editing and re-elongation only work for the case of the 5’→3’ direction
synthesis.
DNA replication in eukaryotes
Processes are quite similar to those in E. coli, but there are several differences.
1. DNA is very long and linear except for those in mitochondria.
2. Multiple replication sites, i.e., multiple oriC.
3. Five different types of polymerases (α, β, γ, δ, ε) are involved.
• α is lagging strand polymerase, and δ is leading strand polymerase of chromosomal DNA.
• γ is mitochondrial DNA polymerase.
• β and ε are repair enzymes.
Replication of mitochondrial DNA polymerase
• DNA are circular and the origins of leading and lagging strands are located at different
places.
1. The synthesis of leading strand is initiated and produced a displacement (D-loop).
2. When the replication of leading strand reaches at the origin of lagging strand, the synthesis
of lagging strand is started on the D-loop.
Reverse transcriptase
In retroviruses, a DNA is synthesized using an RNA as a template.
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Chapter 24 (Summary)
Takusagawa’s Note
1. A host tRNA is attached to the viral RNA as the primer.
2. A single strand DNA is synthesized on the RNA template --- formation of RNA:DNA
hybrid.
3. RNase H degrades the viral RNA.
4. The second DNA strand is synthesized on the first DNA strand.
• RT does not have the editing function (3’→5’ exonuclease). Therefore there is high error
rate in replication, and thus RT has a high mutation rate.
• AZT, ddI, ddC, and 2’3’-didehydro-3’-deoxythymine inhibit the RT activity by stopping the
chain elongation because these nucleotide analogues do not have 3’-OH.
10. Telomeres and telomerase
• In the lagging strand synthesis, a telomere (a short DNA with repeating G rich sequence) is
attached at the 3’-end of chromosomes to complete the DNA synthesis.
• Telomeres are synthesized by telomerase which has a short RNA template (AACCCCAAC).
• Without telomerase action, a chromosome would be shortened with every cycle of DNA
replication and cell division.
• Somatic cells do not have telomerase, thus gradually shortened upon aging, and finally the
cell die. However ~80% of somatic cancer cells have a telomerase function.
11. DNA repair
• DNA is not inert substance, it is dimerized by UV, chemically alkylated, deaminated, and
cleaved its glycosidic bonds.
1. Direct reversal of damage
• Photoreactivating enzymes or DNA photolyases restore the dimerization.
• Some methyltransferases remove methyl groups from O6-alkylguanine.
2. Excision repair
• The damaged section is removed by UvrABC endonuclease. Pol I synthesizes a new DNA on
the template.
• Glycosylases remove the damaged bases, and AP nucleases remove the segments containing
apurinic and apyrimidic residues.
• C residues are naturally deaminated to U residues, and Pol III uses both dTTP and dUTP.
Thus, U can be found in DNA with relatively high rate. U can make base-pair with either A
or G. Therefore this is a serious problem.
• Uracil N-glycosylase recognizes the U rings in DNA and removes them.
3. Recombination repair
• Replication of damaged DNA produces one normal DNA and damaged DNA. A section of
the normal DNA is cut and ligated into the damaged DNA. Then repair is carried out.
4. SOS response (in E. coli)
• Under the normal growth, repair gene SOS (contains recA, uvrA, uvrB genes) is repressed by
LexA proteins.
• LecA proteins activated by single strand DNA produced by DNA damages cleave LexA
repressors and inactivate them. Therefore, SOS repression is released and repair proteins
uvrABC are synthesized.
12. DNA methylation
• m6A, m5C and m4C are the only types of DNA modification in cellular organisms.
• These methyl groups are on the surface of the major groove of DNA, and thus interact with
proteins.
• In some bacteria, methylation protect from restriction enzymes, whereas in eukaryotes m5C
(only occurs) controls transcription. For example, very active globin gene is less methylated;
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Takusagawa’s Note
Chapter 24 (Summary)
specific DNA methylation inhibits its transcription; inhibition of methyltransferases activate
gene expression and protein synthesis.
• Methylation occurs in CG in certain palindromic sequences.
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