Replication

(DNA Synthesis)

Chemistry 40

How Is DNA Replicated?

• DNA replication is semiconservative
• DNA replication is bidirectional
• Replication requires unwinding of the
DNA helix
• DNA replication is semidiscontinuous
• The lagging strand is formed from
Okazaki fragments

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Chemistry 40
Fig. 10-2, p.242

2
 Features of DNA Replication
• DNA replication is bidirectional
– Bidirectional replication involves two
replication forks, which move in opposite
directions
• DNA replication is semi-discontinuous
– The leading strand copies continuously
– The lagging strand copies in segments
(Okazaki fragments) which must be joined

Chemistry 40

Bidirectional
replication of the
E. coli
chromosome. (a)
If replication is
bidirectional, auto-
radiograms of
radioactively
labeled replicating
chromosomes
should show two
replication forks
heavily labeled
with radioactive
thymidine. (b) An
autoradiogram of
the chromosome
from a dividing E.
coli cell shows
bidirectional
replication. (Photo
courtesy of David M.
Prescott, University
of Colorado.)

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 Chemistry 40
Fig. 10-4, p.244

Features of Replication
Mostly in E. coli, but many features are general
• Replication is bidirectional
• The double helix must be unwound - by
helicases
• Supercoiling must be compensated - by DNA
gyrase
• Replication is semidiscontinuous
• Leading strand is formed continuously
• Lagging strand is formed from Okazaki
fragments - discovered by Tuneko and Reiji "O"
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Chemistry 40
Fig. 10-5a, p.245

Chemistry 40
Fig. 10-5b, p.245

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Chemistry 40
Fig. 10-10, p.249

Chemistry 40
Fig. 10-11, p.250

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Chemistry 40

Chemistry 40
Table 10-2, p.242

7
 DNA Polymerase III
The "real" polymerase in E. coli
• At least 10 different subunits
• "Core" enzyme has three subunits - α, ε, and θ
• Alpha subunit is polymerase
• Epsilon subunit is 3'-exonuclease
• Theta subunit is involved in holoenzyme
assembly
• The beta subunit dimer forms a ring around
DNA
• Enormous processivity - 5 million bases!
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8
 Chemistry 40

How Is DNA Replicated in Eukaryotic Cells?

• The cell cycle controls the timing of DNA

replication
• Eukaryotic cells contain a number of different
DNA polymerases
• DNA polymerase d is the principal DNA
replicase

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9
The eukaryotic cell cycle.
The stages of mitosis and
cell division define the M
phase (M for mitosis). G1
(G for gap, not growth) is
typically the longest part of
the cell cycle; G1, is
characterized by rapid
growth and metabolic
activity. Cells that are
quiescent, that is, not
growing and dividing (such
as neurons), are said to be
in G0. The S phase is the
time of DNA synthesis. S
is followed by G2, a
relatively short period of
growth when the cell
prepares for cell division.
Cell cycle times vary from
less than 24 hours (rapidly
dividing cell such as the
epithelial cells lining the
mouth and gut) to
hundreds of days.

How Are the Ends of Chromosomes

Replicated?

• Telomeres, the structures at the ends of

eukaryotic chromosomes, consist of 5-8 bp
tandemly repeated G-rich nucleotide
sequences
• Telomeres are 1-12 kbp long
• Telomeres are replicated by an RNA-
dependent DNA polymerase called
telomerase

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Telomere replication. (a) In replication of the lagging strand, short RNA primers are added
(pink) and extended by DNA polymerase. When the RNA primer at the 5′-end of each strand
is removed, there is no nucleotide sequence to read in the next round of DNA replication.
The result is a gap (primer gap) at the 5′-end of each strand (only one end of the chromosome
is shown in this figure). (b) Asterisks indicate sequences at the 3′-end that cannot be copied
by conventional DNA replication. Synthesis of telomeric DNA by telomerase extends the 5′-
ends of DNA strands, allowing the strands to be copied by normal DNA replication.

Eukaryotic DNA Replication

Like E. coli, but more complex
• Human cell: 6 billion base pairs of DNA to
copy
• Multiple origins of replication: 1 per 3- 300
kbp
• DNA polymerase alpha - four subunits,
polymerase (processivity = 200) but no 3'-
exonuclease
• DNA polymerase beta - similar to alpha

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11
 More Eukaryotic polymerases

• DNA polymerase gamma - DNA-

replicating enzyme of mitochondria
• DNA polymerase delta has a 3'-
exonuclease as well as proliferating cell
nuclear antigen (PCNA)
• PCNA give delta unlimited processivity
and is homologous with prokaryotic pol III
• DNA polymerase epsilon - highly
processive, but does not have a subunit
like PCNA
Chemistry 40

How Are RNA Genomes Replicated?

• Many viruses have genomes composed of

RNA
• DNA is an intermediate in the replication of
RNA viruses
• The viral RNA serves as a template for DNA
synthesis
• The RNA-directed DNA polymerase is called
Reverse Transcriptase

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Figure 28.16
The structures of AZT (3′-azido-2′,3′-
dideoxythymidine). This nucleoside
was the first approved drug for
treatment of AIDS. AZT is
phosphorylated in vivo to give
AZTTP (AZT 5′-triphosphate), a
substrate analog that binds to HIV
reverse transcriptase, HIV reverse
transcriptase incorporates AZTTP
into growing DNA chains in place of
dTTP. Incorporated AZTMP blocks
further chain elongation because its
3′-azido group cannot form a
phosphodiester bond with an
incoming nucleotide. Host cell DNA
polymerases have little affinity for
AZTTP.

Reverse Transcriptase
• Primer required, but a strange one - a tRNA
molecule that the virus captures from the host
• RT transcribes the RNA template into a
complementary DNA (cDNA) to form a
DNA:RNA hybrid
• All RNA tumor viruses contain a reverse
transcriptase

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13
 Reverse Transcriptase Activities
• Three enzyme activities
– RNA-directed DNA polymerase
– RNase H activity - degrades RNA in the
DNA:RNA hybrids
– DNA-directed DNA polymerase - which makes
a DNA duplex after RNase H activity destroys
the viral genome
• HIV therapy: AZT (or 3'-azido-2',3'-
dideoxythymidine) specifically inhibits RT

Chemistry 40

Chemistry 40
Table 10-3, p.250

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