Transcript CHAPTER 3 DNA Replication

Peter J. Russell
A molecular Approach 2nd Edition
CHAPTER 3
DNA Replication
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy, 台大農藝系
NTU
遺傳學 601 20000
Chapter 3 slide 1
Semiconservative DNA Replication
1. Watson and Crick DNA model implies a
mechanism for replication:
a. Unwind the DNA molecule.
b. Separate the two strands.
c. Make a complementary copy for each strand.
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Chapter 3 slide 2
2 .Three possible models were proposed for DNA
replication:
a. Conservative model proposed both strands of one copy would be
entirely old DNA, while the other copy would have both strands
of new DNA.
b. Dispersive model was that dsDNA might fragment, replicate
dsDNA, and then reassemble, creating a mosaic of old and new
dsDNA regions in each new chromosome.
c. Semiconservative model is that DNA strands separate, and a
complementary strand is synthesized for each, so that sibling
chromatids have one old and one new strand. This model was the
winner in the Meselson and Stahl experiment.
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Chapter 3 slide 3
Fig. 3.1 Three models for the replication of DNA
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Chapter 3 slide 4
The Meselson-Stahl Experiment
Animation: The Meselson-Stahl Experiment
1. Meselson and Stahl (1958) grew E. coli in a heavy (not
radioactive) isotope of nitrogen, 15N in the form of
15NH Cl. Because it is heavier, DNA containing 15N is
4
more dense than DNA with normal 14N, and so can be
separated by CsCl density gradient centrifugation (Box
3.1).
2. Once the E. coli were labeled with heavy 15N, the
researchers shifted the cells to medium containing normal
14N, and took samples at time points. DNA was extracted
from each sample and analyzed in CsCl density gradients
(Figure 3.2).
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Chapter 3 slide 5
Fig. 3.2 The Meselson-Stahl experiment, which showed that DNA replicates
semiconservatively
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Chapter 3 slide 6
Box Fig. 3.1 Equilibrium centrifugation of DNA of different densities in a cesium chloride
density gradient
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Chapter 3 slide 7
3. After one replication cycle in normal 14N medium, all DNA had density
intermediate between heavy and normal. After two replication cycles,
there were two bands in the density gradient, one at the intermediate
position, and one at the position for DNA containing entirely 14N.
4. Results compared with the three proposed models:
a. Does not fit conservative model, because after one generation there is a
single intermediate band, rather than one with entirely 15N DNA and
another with entirely 14N DNA.
b. The dispersive model predicted that a single band of DNA of
intermediate density would be present in each generation, gradually
becoming less dense as increasing amounts of 14N were incorporated
with each round of replication. Instead, Meselson and Stahl observed
two bands of DNA, with the intermediate form decreasing over time.
c. The semiconservative model fits the data very well.
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Chapter 3 slide 8
Semiconservative DNA Replication in
Eukaryotes
1. To visualize DNA of
eukaryotic chromosomes
replicating, CHO
(Chinese hamster ovary)
cells are grown in 5bromodeoxyuridine
(BUdR), a base analog for
thymine. After two rounds
of replication, mitotic
chromosomes are stained
with fluorescent dye and
Giemsa stain (Figure 3.3).
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Chapter 3 slide 9
2. DNA containing T stains darkly, while DNA containing
two BUdR strands stains lightly. Observed that after one
generation, both chromatids stain the same, each with one
BUdR strand and one T strand. After two generations,
they stain differently and are called harlequin
chromosomes, one light (both strands have BUdR) and
one dark (one strand has BUdR and other strand has T).
3. Showed that eukaryotes also use semiconservative DNA
replication.
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Chapter 3 slide 10
DNA Polymerases, the DNA Replicating
Enzymes
1. First isolation of an enzyme involved in DNA
replication was in 1955. Arthur Kornberg won the
1959 Nobel Prize in Physiology or Medicine for
this work.
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Chapter 3 slide 11
DNA Polymerase I
1. Accomplished in vitro synthesis of E. coli DNA. His
reaction mixture included:
a. DNA fragments (template).
b. Radioactively labeled dNTPs (dATP, dGTP, dTTP and dCTP).
c. E. coli lysate.
2. Enzyme originally called the Kornberg enzyme, now
known as DNA Polymerase I. Once isolated, could
characterize its activity, showing that the above
components are required, along with Mg2+ ions for
maximum activity.
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Chapter 3 slide 12
Roles of DNA Polymerases
Animation: DNA Biosynthesis: How a New DNA Strand is Made
1. All DNA polymerases link dNTPs into DNA chains (Figure 3.4). Main
features of the reaction:
a. An incoming nucleotide is attached by its 5’-phosphate group to the 3’OH of the growing DNA chain. Energy comes from the dNTP releasing
two phosphates. The DNA chain acts as a primer for the reaction.
b. The incoming nucleotide is selected by its ability to hydrogen bond
with the complementary base in the template strand. The process is fast
and accurate.
c. DNA polymerases synthesize only from 5’ to 3’.
2. The enzyme Kornberg isolated was believed to be the only DNA
polymerase in E. coli. However, mutations in this gene (polA1) were
not lethal, indicating that other DNA polymerases must exist in E. coli.
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Chapter 3 slide 13
Fig. 3.4a DNA chain elongation catalyzed by DNA polymerase
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Chapter 3 slide 14
Fig. 3.4b DNA chain elongation catalyzed by DNA polymerase
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Chapter 3 slide 15
3. Temperature-sensitive mutants are used to study
essential genes. At 37°C polAex1 strains are
normal, and the protein has normal activity in
vitro. At 42°C, however, the protein lacks 5’-3’
exonuclease activity, and bacterial cells with this
mutation are dead.
4. Additional DNA polymerases have been isolated,
including DNA polymerase II (1970), DNA
polymerase III (1971), DNA polymerase IV, and
DNA polymerase V.
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Chapter 3 slide 16
The properties of DNA polymerases
5. The properties of known E. coli DNA polymerases are:
a. DNA polymerase I is a single peptide encoded by polA and used for DNA
replication. Replicates DNA in the 5’3’ direction. Has 5’3’
exonuclease activity to remove nucleotides from 5’ end of DNA or from an
RNA primer.
b. DNA polymerase II is a single peptide encoded by polB. Used for DNA
repair.
c. DNA polymerase III has three polypeptide subunits in the catalytic core of
the enzyme: α (encoded by the dnaE gene), ε(dnaQ), and θ(holE).
Holoenzyme has an additional six different polypeptides. Replicates DNA
in the 5’3’ direction.
d. DNA polymerase IV is encoded by the dinB gene, and is used in DNA
repair.
e. DNA polymerase V is encoded by umuDC, and is used in DNA repair.
6. E. coli DNA polymerases used for DNA replication (DNA polymerase I
and DNA polymerase III) have 3’5’ exonuclease (proofreading)
activity.
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Chapter 3 slide 17
Molecular Model of DNA Replication
1. Table 3.1 shows key genes and DNA sequences
involved in replication.
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Chapter 3 slide 18
Initiation of Replication
1. Replication starts when DNA at the origin of replication denatures to
expose the bases, creating a replication fork. Replication is usually
bidirectional from the origin. E. coli has one origin, oriC, which has:
a. A minimal sequence of about 245 bp required for initiation.
b. Three copies of a 13-bp AT-rich sequence.
c. Four copies of a 9-bp sequence.
2. Events in E. coli initiating DNA synthesis, derived from in vitro studies
(Figure 3.5):
a. Initiator proteins attach. E. coli’s initiator protein is DnaA (from the
dnaA gene).
b. DNA helicase (from dnaB) binds initiator proteins on the DNA, and
denatures the AT-rich region using ATP as an energy source.
c. DNA primase (from dnaG) binds helicase to form a primosome, which
synthesizes a short (5–10nt) RNA primer. .
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Chapter 3 slide 19
Fig. 3.5 Model for the formation of a replication bubble at a replication origin in
E. coli and the initiation of the new DNA strand
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Chapter 3 slide 20
Semidiscontinuous DNA Replication
• Animation: Molecular Model of DNA Replication
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Chapter 3 slide 21
Fig. 3.6a, b Model for the events occurring around a single replication fork of the
E. coli chromosome
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Chapter 3 slide 22
Fig. 3.6c-e Model for the events occurring around a single replication fork of the
E. coli chromosome
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 3 slide 23
1. When DNA denatures at the oriC, replication forks are
formed. DNA replication is usually bi-directional, but will
consider events at just one replication fork (Figure 3.6):
a. Single-strand DNA-binding proteins (SSBs) bind the ssDNA
formed by helicase, preventing reannealing.
b. Primase synthesizes a primer on each template strand.
c. DNA polymerase III adds nucleotides to the 3’ end of the primer,
synthesizing a new strand complementary to the template, and
displacing the SSBs. DNA is made in opposite directions on the
two template strands.
d. New strand made 5’ → 3’ in same direction as movement of the
replication fork is leading strand, while new strand made in
opposite direction is lagging strand. Leading strand needs only
one primer, while lagging needs a series of primers.
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Chapter 3 slide 24
2. Helicase denaturing DNA causes tighter winding in other
parts of the circular chromosome. Gyrase relieves this
tension.
3. Leading strand is synthesized continuously, while lagging
strand is synthesized discontinuously, in the form of
Okazaki fragments. DNA replication is therefore
semidiscontinuous.
4. Each fragment requires a primer to begin, and is extended
by DNA polymerase III.
5. Okazaki data show that these fragments are gradually joined
together to make a full-length dsDNA chromosome. DNA
polymerase I uses the 3’-OH of the adjacent DNA fragment
as a primer, and simultaneously removes the RNA primer
while resynthesizing the primer region in the form of DNA.
The nick remaining between the two fragments is sealed
with DNA ligase. (Fig. 3.7)
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Chapter 3 slide 25
Fig. 3.7 Action of DNA ligase in sealing the gap between adjacent DNA fragments to
form a longer, covalently continuous chain
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Chapter 3 slide 26
6. Key proteins are associated to form a replisome.
Template DNA probably bends to allow synthesis
of both leading and lagging strands at the
replication fork (Fig. 3.8)
7. Early stages of bidirectional replication are
summarized in Figure 3.9.
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Chapter 3 slide 27
Fig. 3.8 Model for the “replication machine,” or replisome, the complex of key
replication proteins, with the DNA at the replication fork
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Chapter 3 slide 28
• iActivity: Unraveling DNA Replication
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Chapter 3 slide 29
Fig. 3.9 Bidirectional replication of circular DNA molecules
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Chapter 3 slide 30
Replication of circular DNA and the supercoiling
problem
1. Some circular chromosomes (e.g., E. coli) are
circular throughout replication, creating a thetalike (θ) shape. As the strands separate on one side
of the circle, positive supercoils form elsewhere
in the molecule. Replication fork moves about
500 nt/ second, so at 10 bp/turn, replication fork
rotates at 3,000 rpm.
2. Topoisomerases relieve the supercoils, allowing
the DNA strands to continue separating as the
replication forks advance.
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Chapter 3 slide 31
Fig. 3.11 Diagram showing the unreplicated, supercoiled parent strands and the
portions already replicated
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Chapter 3 slide 32
Rolling Circle Replication
1. Another model for replication is rolling circle(Figure
3.10), which is used by several bacteriophages, including
ΦX174 (after a complement is made for the genomic
ssDNA) and λ (after circularization by base pairing
between the “sticky” ssDNA cos ends)
2. Rolling circle replication begins with a nick (singlestranded break) at the origin of replication. The 5’ end is
displaced from the strand, and the 3’ end acts as a primer
for DNA polymerase III, which synthesizes a continuous
strand using the intact DNA molecule as a template.
3. The 5’ end continues to be displaced as the circle “rolls”,
and is protected by SSBs until discontinuous DNA
synthesis makes it a dsDNA again.
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Chapter 3 slide 33
Fig. 3.10 The replication process of double-stranded circular DNA molecules through
the rolling circle mechanism
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Chapter 3 slide 34
4. A DNA molecule many genomes in length can be made
by rolling circle replication. During viral assembly it is
cut into individual viral chromosomes and packaged into
phage head.
5. Bacteriophage λ, regardless of whether entering the lytic
or lysogenic pathway, circularizes its chromosome
immediately after infection.
a. In a lysogenic infection, the circular DNA integrated into a
specific site in the E. coli chromosome by a crossover event.
b. In a lytic infection, rolling circle replication produces a long
concatamer of λ DNA, and the a viral endonuclease (product of
the ter gene) recognize the cos sites and makes the staggered
cuts that used to assemble new virus particles.
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Chapter 3 slide 35
Fig. 3.11  chromosome structure varies at stages of lytic infection of E. coli
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Chapter 3 slide 36
DNA Replication in Eukaryotes
1. DNA replication is very similar in both
prokaryotes and eukaryotes, except that
eukaryotes have more than one chromosome.
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Chapter 3 slide 37
Replicons
1. Eukaryotic chromosomes generally contain much
more DNA than those of prokaryotes, and their
replication forks move much more slowly. If they
were like typical prokaryotes, with only one origin
of replication per chromosome, DNA replication
would take many days.
2. Instead, eukaryotic chromosomes contain multiple
origins, at which DNA denatures and replication
then proceeds bidirectionally until an adjacent
replication fork is encountered. The DNA replicated
from a single origin is called a replicon, or
replication unit (Figure 3.12).
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Chapter 3 slide 38
Fig. 3.12 Replicating DNA of Drosophila melanogaster
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Chapter 3 slide 39
3. In eukaryotes, replicon size is smaller than it is in
prokaryotes, replication is slower, and each
chromosome contains many replicons. Number and
size of replicons vary with cell type.
4. Not all origins within a genome initiate DNA
synthesis simultaneously. Cell-specific patterns of
origin activation are observed, so that chromosomal
regions are replicated in a predictable order in each
cell cycle (Figure 3.13).
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Chapter 3 slide 40
Fig. 3.13 Temporal ordering of DNA replication initiation events in replication units
of eukaryotic chromosomes
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Chapter 3 slide 41
Initiation of Replication
1. Eukaryotic origins are generally not well characterized; those of the
yeast Saccharomyces cerevisiae are among the best understood.
2. Chromosomal DNA fragments (about 100bp) that are able to replicate
autonomously when introduced into yeast as extracellular, circular
DNA are known as ARSs (autonomously replicating sequences).
3. ARSs are yeast replicators. The three sequence elements typically
found in ARSs are A, B1, and B2.
4. Initiator protein in yeasts is the multiprotein origin recognition complex
(ORC), which binds to A and B1. Other replication proteins join,
including one that unwinds DNA at B2. The yeast origin of replication
is between regions B1 and B2.
5. DNA and histones must be doubled in each cell cycle. G1 prepares the
cell for DNA replication, chromosome duplication occurs during S
phase, G2 prepares for cell division, and segregation of progeny
chromosomes occurs during M phase, allowing the cell to divide.
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Chapter 3 slide 42
6. Cell cycle control is complex, and only outlined here.
Yeasts, in which chromosomal replication is well studied,
serve as a eukaryotic model organism.
7. Initiation of replication has two separate steps, controlled
by cyclin-dependent kinases (Cdks) that are present
throughout the cell cycle, except during G1.
a. In the absence of Cdsk during G1, replicator selection occurs.
ORC and other proteins assemble on each replicator to from prereplicative complexes (pre-RC).
b. When cell enters S phase, Cdks are present, and activate pre-RCs
to initiate replication.
c. Cdk activity inhibits another round of pre-RC formation until the
cell again enters G1, when Cdks are absent.
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Chapter 3 slide 43
Fig. 3.x Some of the molecular events that control progression through the cell cycle
in yeasts
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Chapter 3 slide 44
Eukaryotic Replication Enzymes
1. Enzymes of eukaryotic DNA replication aren’t as well
characterized as their prokaryotic counterparts. The
replication process is similar in both groups—DNA
denatures, replication is semiconservative and
semidiscontinuous and primers are required.
2. Fifteen DNA polymerases are known in mammalian cells:
a. Three DNA polymerases are used to replicate nuclear DNA. Pol α
(alpha) extends the 10-nt RNA primer by about 30nt. Pol δ(delta)
and Pol ε(epsilon) extend the RNA/DNA primers, one on the
leading strand and the other on the lagging (it is not clear which
synthesizes which).
b. Other DNA pols replicate mitochondrial or chloroplast DNA, or are
used in DNA repair.
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Chapter 3 slide 45
Replicating the Ends of Chromosomes
1. When the ends of chromosomes are replicated
and the primers are removed from the 5’ ends,
there is no adjacent DNA strand to serve as a
primer, and so a single-stranded region is left at
the 5’ end of the new strand. If the gap is not
addressed, chromosomes would become shorter
with each round of replication (Figure 3.14).
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Chapter 3 slide 46
Fig. 3.14 The problem of replicating completely a linear chromosome in eukaryotes
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Chapter 3 slide 47
2. Most eukaryotic chromosomes have short, species-specific sequences
tandemly repeated at their telomeres. Blackburn and Greider have
shown that chromosome lengths are maintained by telomerase, which
adds telomere repeats without using the cell’s regular replication
machinery.
3. In the ciliate Tetrahymena, the telomere repeat sequence is 5
TTGGGG-3 (Figure 3.15).
a. Telomerase, an enzyme containing both protein and RNA, binds to the
terminal telomere repeat when it is single stranded, synthesizing a 3-nt
sequence, TTG.
b. The 3 end of the telomerase RNA contains the sequence AAC, which
binds the TTG positioning telomerase to complete its synthesis of the
TTGGGG telomere repeat.
c. Additional rounds of telomerase activity lengthen the chromosome by
adding telomere repeats.
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Chapter 3 slide 48
4. After telomerase adds telomere sequences, chromosomal replication
proceeds in the usual way. Any shortening of the chromosome ends is
compensated by the addition of the telomere repeats.
5. If the sequence of the telomerase RNA is mutated, telomeres will
correspond to the mutant sequence, rather than the organism’s normal
telomere sequence. Using an RNA template to make DNA, telomerase
functions as a reverse transcriptase called TERT (telomerase reverse
transcriptase).
6. Telomere length may vary, but organisms and cell types have
characteristic telomere lengths. Mutants affecting telomere length have
been identified, and data indicate that telomere length is genetically
controlled. Shortening of telomeres eventually leads to cell death,
and this may be a factor in the regulation of normal cell death.
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Chapter 3 slide 49
Fig. 3.15 Synthesis of telomeric DNA by telomerase
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Chapter 3 slide 50
Assembling New DNA into Nucleosomes
1. When eukaryotic DNA is replicated, it complexes with histones. This
requires synthesis of histone proteins and assembly of new nucleosomes.
2. Transcription of histone genes is initiated near the end of G1 phase, and
translation of histone proteins occurs throughout S phase.
3. Assembly of newly replicated DNA into nucleosomes is shown in Figure
3.16.
a. Parental histone cores separate into an H3-H4 tetramer, and two H2A-H2B
dimers.
b. H3-H4 tetramer (preexisting or newly made) binds to replicated dsDNA
and begins nucleosome assembly.
c. H2A-H2B dimers (preexisting or newly made) are added in an assembly
process that requires histone chaperone proteins to direct it.
4. Self-assembly of nucleosomes has been observed only in vitro.
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Chapter 3 slide 51
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Chapter 3 slide 52