The Replication of DNA
THE CHEMISTRY OF DNA SYNTHESIS
DNA synthesis requires dNTPs and a primer:template
junction
(Polarity of primer is important)
The addition of a deoxyribonucleotide
to the 3' end of a polynucleotide chain
(the primer strand) is the fundamental
reaction by which DNA is synthesized.
As shown, base-pairing between an
incoming deoxyribonucleoside
triphosphate and an existing strand of
DNA (the template strand) guides the
formation of the new strand of DNA
and causes it to have a complementary
nucleotide sequence
(Which p is incorporated into
the new chain?)
DNA is synthesized by extending the 3’end of the primer
Hydrolysis of pyrophosphate is the driving force for
DNA Synthesis
G= -7
kcal/mole
THE MECHANISM OF DNA POLYMERASE
DNA polymerases use a single active site to catalyze
DNA synthesis
Correctly paired bases are required
Steric constrains preventing DNA polymerase from using rNTP
precursors
Incorporation assays can be used
to measure DNA synthesis
Anticancer and antiviral agents target DNA
replications
DNA polymerases resemble a hand that grips the
primer:template junction
Two metal ions bound to DNA polymerase catalyze the nucleotide
addition
DNA polymerase “grips” the template and the incoming
nucleotide when a correct base pair is made
The path of the template DNA through the DNA polymerase; note
the bend in the template between the first and second bases
DNA polymerases are processive enzymes; thus the rate
of DNA synthesis is dramatically increased (~1000 bp/sec)
The thumb helps to
maintain a strong
association between the
DNA polymerase and its
substrate
Exonucleases proofread newly synthesized DNA
THE REPLICATION FORK
Two replication forks moving in
opposite directions on a circular
chromosome.
An active zone of DNA
replication moves progressively
along a replicating DNA
molecule, creating a Y-shaped
DNA structure known as a
replication fork: the two arms of
each Y are the two daughter DNA
molecules, and the stem of the Y
is the parental DNA helix. In this
diagram, parental strands are
orange; newly synthesized
strands are red.
Both strands of DNA are synthesized together at the
replication fork
DNA synthesis proceeds in a 5’ to 3’ direction and
is semi-discontinuous.
The structure of a DNA replication fork.
Because both daughter DNA strands are polymerized in the 5'to-3'
direction, the DNA synthesized on the lagging strand must be made
initially as a series of short DNA molecules, called Okazaki fragments
The initiation of a new strand of DNA requires an RNA
primer
A Special Nucleotide-Polymerizing Enzyme Synthesizes
Short RNA Primer Molecules on the Lagging Strand
RNA primer synthesis. A
schematic view of the reaction
catalyzed by DNA primase, the
enzyme that synthesizes the
short RNA primers made on
the lagging strand using DNA
as a template. Unlike DNA
polymerase, this enzyme can
start a new polynucleotide
chain by joining two or three
nucleoside triphosphates
together. The primase
synthesizes a short
polynucleotide in the 5'-to-3'
direction and then stops,
making the 3' end of this
primer available for the DNA
polymerase
RNA primers must be removed
to complete DNA replication
DNA helicases unwind the double helix in advance of
replication fork
An assay used to test for DNA helicase
enzymes.
A short DNA fragment is annealed to a
long DNA single strand to form a region
of DNA double helix. The double helix
is melted as the helicase runs along the
DNA single strand, releasing the short
DNA fragment in a reaction that requires
the presence of both the helicase protein
and ATP. The rapid step-wise movement
of the helicase is powered by its ATP
hydrolysis
Biochemical assay for DNA helicase activity
DNA helicase polarity
The structure of a DNA helicase.
(A) A schematic diagram of the protein as a hexameric ring. (B) Schematic diagram
showing a DNA replication fork and helicase to scale. (C) Detailed structure of the
bacteriophage T7 replicative helicase, as determined by x-ray diffraction. Six identical
subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule along a
DNA single strand that passes through the central hole. Red indicates bound ATP
molecules in the structure.
DNA helicase pulls single-stranded DNA through a
central protein pore
Single-stranded DNA-binding proteins (SSB) stabilize
ssDNA prior to replication
The effect of single-strand DNA-binding proteins (SSB proteins) on the structure of singlestranded DNA.
Because each protein molecule prefers to bind next to a previously bound molecule, long rows of
this protein form on a DNA single strand. This cooperative binding straightens out the DNA
template and facilitates the DNA polymerization process. The “hairpin helices” shown in the
bare, single-stranded DNA result from a chance matching of short regions of complementary
nucleotide sequence; they are similar to the short helices that typically form in RNA molecules
Topoisomerases remove supercoils produced by DNA
unwinding at the replication fork
Replication fork enzymes extend the range of DNA
polymerase
THE SPECILIZATION OF DNA POLYMERASES
DNA polymerases are specialized for different roles in the
cell
DNA polymerase switching during eukaryotic DNA replication
Sliding clamps dramatically increase DNA polymerase
processivity
Sliding clamps of E. coli, T4 and eukaryotic cells
Sliding clamps are opened and placed on DNA by clamp
loaders
DNA SYNTHESIS AT THE REPLICATION
FORK
Composition of the DNA pol III holoenzyme (E. coli)
E. Coli replication fork
08_Figure22a.jpg
08_Figure22b.jpg
08_Figure22c.jpg
08_Figure22d.jpg
08_Figure22e.jpg
Interactions between replication fork proteins from
the E. coli replisome
INITIATION OF DNA REPLICATION
Specific genomic DNA sequences direct the initiation
of DNA Replication
The replicon model of
replication initiation
Replicator sequences (origin of replication) include
initiator binding sites and easily unwound DNA
BINDING AND UNWINDING: ORIGIN SELECTION
AND ACTIVATION BY THE INITIATOR PROTEIN
Two replication forks moving in
opposite directions on a circular
chromosome.
An active zone of DNA replication
moves progressively along a
replicating DNA molecule, creating a
Y-shaped DNA structure known as a
replication fork: the two arms of each
Y are the two daughter DNA
molecules, and the stem of the Y is
the parental DNA helix. In this
diagram, parental strands are orange;
newly synthesized strands are red.
The identification of origins of replication
Genetic identification of replicators (origins)
08_UnFigure09.jpg
Protein-protein and protein-DNA interactions direct the
initiation process
Initiation of DNA Replication (E. coli)
Eukaryotic chromosomes are replicated exactly once per
cell cycle
Incomplete replication causes chromosome
breakage
Replicators are inactivated by DNA replication
Prereplicative complex formation is the first step in the
initiation of replication in eukaryotes
Assembly of the eukaryotic replication fork
Pre-RC formation and activation are regulated to allow
only a single round of replication during each cell cycle
Cdks: cyclin-dependent kinases
Cell cycle regulation of Cdk activity and pre-RC formation
Similarities between eukaryotic and prokaryotic DNA
replication initiation
FINISHING REPLICATION
Type II topoisomerases are required
to separate daughter DNA molecules
Lagging-strand synthesis is unable to copy the extreme
ends of linear chromosomes
One solution of the end problem is to use protein priming
Telomerase Replicates the Ends of Eukaryotic
Chromosomes
The structure of telomerase.
The telomerase is a protein–
RNA complex that carries an
RNA template for synthesizing
a repeating, G-rich telomere
DNA sequence. Only the part of
the telomerase protein
homologous to reverse
transcriptase is shown here
(green). A reverse transcriptase
is a special form of polymerase
enzyme that uses an RNA
template to make a DNA strand;
telomerase is unique in carrying
its own RNA template with it at
all times.
Replication of telomeres by telamerase
Telamerase solves the end problem by extending the 3’
end of the chromosome
Telomere-binding proteins regulate telomerase activity
and telomere length
S. cerevisiae
In human, these proteins form a complex called
“Shelterin” to shelter telomeres from DNA repair
enzymes
Telomeres form a looped structure in the cell
The Nobel Prize in Physiology or Medicine 2009
"for the discovery of how chromosomes are protected by
telomeres and the enzyme telomerase"
Elizabeth H. Blackburn
Carol W. Greider
Jack W. Szostak
1/3 of the prize
1/3 of the prize
1/3 of the prize
USA
USA
USA
University of California
San Francisco, CA, USA
b. 1948
(in Hobart, Tasmania,
Johns Hopkins University
School of Medicine
Baltimore, MD, USA
b. 1961
Harvard Medical School;
Massachusetts General
Hospital
Boston, MA, USA; Howard
Hughes Medical Institute
b. 1952
(in London, United Kingdom)
Is This The Fountain Of Youth?
Scientists Find Way to Partially Reverse Aging in
Mice
Scientists reverse some age effects in mice Researchers artificially age
rodents by suppressing a gene that helps repair telomeres, then rejuvenate
them by turning back on the genetic switch. But the work is far from any use
on humans. (Dr. Ronald DePinho, a molecular biologist at the Dana-Farber
Cancer Institute at Harvard Medical School), Los Angeles Times
Telomerase in human
Active
Down
Downregulated
regulated
Telomerase is on during fetal
development and remain
active in various proliferative
cells.
- stem cell, germ cell, hair,
activated lymphocytes, etc
Telomerase is down-regulated
but still detectable in many
other adult cells.
-epithelial
-fibroblast
-endothelial
Active
Highly active
Active
Highly
90% of human tumors
iBioSeminars: Elizabeth
Blackburn, June 2008
Purified DNA can be labeled with radioactive or
chemical markers
Methods for labeling DNA molecules in vitro.
(A) A purified DNA polymerase enzyme labels all the
nucleotides in a DNA molecule and can thereby produce
highly radioactive DNA probes. (B) Polynucleotide
kinase labels only the 5' ends of DNA strands; therefore,
when labeling is followed by restriction nuclease
cleavage, as shown, DNA molecules containing a single
5'-end-labeled strand can be readily obtained. (C) The
method in (A) is also used to produce nonradioactive
DNA molecules that carry a specific chemical marker that
can be detected with an appropriate antibody. The
modified nucleotide shown can be incorporated into DNA
by DNA polymerase so as to allow the DNA molecule to
serve as a probe that can be readily detected. The base on
the nucleoside triphosphate shown is an analog of
thymine in which the methyl group on T has been
replaced by a spacer arm linked to the plant steroid
digoxigenin. To visualize the probe, the digoxigenin is
detected by a specific antibody coupled to a visible
marker such as a fluorescent dye. Other chemical labels
such as biotin can be attached to nucleotides and used in
essentially the same way.
DNA hybridization can be used to identify specific
DNA molecules
In situ hybridization to locate
specific genes on chromosomes.
Here, six different DNA probes have
been used to mark the location of their
respective nucleotide sequences on
human chromosome 5 at metaphase.
The probes have been chemically
labeled and detected with fluorescent
antibodies. Both copies of
chromosome 5 are shown, aligned side
by side. Each probe produces two dots
on each chromosome, since a
metaphase chromosome has replicated
its DNA and therefore contains two
identical DNA helices.
Hybridization probes can identify electrophoretically
separated DNA and RNAs
Northern and Southern blotting facilitate hybridization with
electrophoretically separated nucleic acid molecules
Southern Blots
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•
•
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Another way to find desired fragments
Subject the DNA library to agarose gel
electrophoresis
Soak gel in NaOH to convert dsDNA to ssDNA
Neutralize and blot gel with nitrocellulose sheet
Nitrocellulose immobilizes ssDNA
Incubate sheet with labeled oligonucleotide probes
Autoradiography should show location of desired
fragment(s)
PCR (polymerase chain reaction) amplifies DNAs by
repeated rounds of DNA replication in vitro
Gene can be selectively amplified by PCR
DNA fingerprinting
The Nobel Prize in Chemistry 1993 was awarded "for contributions
to the developments of methods within DNA-based chemistry" jointly
with one half to Kary B. Mullis "for his invention of the polymerase
chain reaction (PCR) method"and with one half to Michael
Smith "for his fundamental contributions to the establishment of
oligonucleotide-based, site-directed mutagenesis and its
development for protein studies".
How to determine DNA sequences?
ddNTPs used in DNA sequencing
Chain termination in the presence of ddNTPs
DNA sequencing by chain-termination method
DNA sequencing gel
Automated DNA sequencing.
Shown here is a tiny part of the data from an automated DNA-sequencing run as it
appears on the computer screen. Each colored peak represents a nucleotide in the DNA
sequence—a clear stretch of nucleotide sequence can be read here between positions
173 and 194 from the start of the sequence. This particular example is taken from the
international project that determined the complete nucleotide sequence of the genome of
the plant Arabidopsis.
The Nobel Prize in Chemistry 1980 was divided, one half awarded
to Paul Berg "for his fundamental studies of the biochemistry of
nucleic acids, with particular regard to recombinant-DNA",the
other half jointly to Walter Gilbert and Frederick Sanger "for their
contributions concerning the determination of base sequences in
nucleic acids".
Photos: Copyright © The Nobel Foundation
Genomic sequences provide the
ultimate genetic libraries
The human genomic project strategy
Shotgun sequencing a bacterial genome then a partial
assembly of large genome sequences
Contigs are linked by sequencing the ends of large DNA fragments
The pair-end strategy permits
the assembly of large-genome
scaffolds
The Human Genome Project, which
unveiled its landmark results a decade
ago, relied on the laborious Sanger
sequencing method. This involves
building complementary DNA strands
to match the original sample, until
nucleotides labelled with a fluorescent
dye are added to halt the process. The
copied fragments are then sorted by
size to determine the sequence of the
original strand.
More recently, faster 'next generation' techniques were developed to
read a DNA sequence by tracking the construction of a complementary
strand as it actually happens. Most methods use fluorescent labelling to
identify individual nucleotides as they are added. But these reagents are
expensive — each sequencing run can cost thousands of dollars, and
may still take more than a week to complete.
The $500 human genome is within reach
Ion Torrent's device instead uses cheaper, natural nucleotides, and
senses the hydrogen ions (protons) that are released as each
nucleotide is incorporated onto the complementary DNA.
Ion Personal Genome Machine (PGM)
Each Ion Torrent chip sports 1.2 million DNA-testing wells
Microscopic beads carrying fragments of DNA are first loaded into 1.2 million 3.5micrometre-wide wells covering a small chip that cost $99. The chip is then flooded
with washes of different nucleotides bearing the four bases that make up DNA, one
after another. The wells are cleaned between each wash. If a nucleotide is
complementary to the next unpaired base on the bead, it binds and gives off a
hydrogen ion, changing the pH inside the well. This produces an electrical signal,
indicating that the base in that particular wash is the next letter of the sequence.
Each step takes less than five seconds, enabling a single chip to read about 25
million bases in a single two-hour run, and for just a few hundred dollars.
(Nature 475: 348-352, 2011)