Download DNA Polymerase I

Chapter 10
Replication of DNA
© John Wiley & Sons, Inc.
Chapter Outline
Basic Features of DNA Replication In Vivo
DNA Replication in Prokaryotes
Unique Aspects of Eukaryotic DNA
Replication
© John Wiley & Sons, Inc.
Basic Features of DNA Replication In Vivo
DNA replication occurs semiconservatively, is initiated at unique
origins, and usually proceeds bidirectionally from each origin of
replication.
Synthesis of DNA (RNA,proteins):
1-initiation, 2-extension/elongation, 3-termiantion.
DNA polymerase (protein-enzyme)-essential for conservation of
any species
3,000/30,000 nucleotides per minutes
One mistake per billion of nucleotides
© John Wiley & Sons, Inc.
DNA Replication is Semiconservative
MODEL
 Each strand serves as a
template
 Complementary base
pairing determines the
sequence of the new
strand
 Each strand of the
parental helix is
conserved
 Semiconservative=half
conserve
© John Wiley & Sons, Inc.
Possible Models of
DNA Replication
© John Wiley & Sons, Inc.
CsCl Equilibrium Density-Gradient Centrifugation
Bacteria growing with 15N and 14N (normal)
Density:
GsCl
~1.7 g/cm3
DNA
~1.710 g/cm3 with 14N
~1.724 g/cm3 with 15N
Bacteria with 15N-parental
(several periods of time)
Bacteria with 14N-daugther
Centrifugation:
process involving the centrifugal force for the
sedimentation of particles and/or molecules
[revolutions per minute (RPM)]
gravitational force to cause precipitation/sedimentation
© John Wiley & Sons, Inc.
The Meselson-Stahl Experiment:
DNA Replication in E. coli is Semiconservative
Bacteria growing with 15N for several generations
Change medium and add 14N
--one generation
--two generations
--three generations
© John Wiley & Sons, Inc.
Semiconservative Replication in Eukaryotes
Autoradiography: A technique using
X- ray film to visualize molecules or
fragments of molecules that have been
radioactively labeled
1H-Thymidine
(normal)
3H-Thymidine
Autoradiography
3H=tritium
© John Wiley & Sons, Inc.
3H-Thymidine
1H-Thymidine
C-metaphase: Colchicine-metaphase:
Colchincine: is a toxic natural product and secondary metabolite
and it inhibits microtubule© John
polymerization
by binding to tubulin.
Wiley & Sons, Inc.
The Origin of Replication in E. coli
Replicon: is a sequence of DNA at which DNA replication is initiated on a chromosome,
plasmid or virus.
-OriC (245 bp)
-AT-rich region (replication
bubble)
-13-mer and 9-mer tandem
N: any nucleotide
Eukaryotic:
ARS
(Autonomously Replicating sequences)
AT-rich region 11bp
Mer=repeating unit=parts
© John Wiley & Sons, Inc.
Bidirectional Replication of the
Circular E. coli Chromosome
-Circular DNA (double strand DNA)
--Unwind (access and single strand DNA)
--Simultaneous semiconservative replication
--Swivel (point of break) Topoisomerases
--Y-shape structure=replication fork
Topoisomerases: are enzymes that regulate the overwinding or underwinding of DNA.
© John Wiley & Sons, Inc.
Visualization of Replication in E. coli
3H-Thymidine
Autoradiography
© John Wiley & Sons, Inc.
Visualization of Replication in E. coli
3H-Thymidine
Autoradiography
© John Wiley & Sons, Inc.
Bidirectional Replication: The Phage Chromosome
-Small bacterial virus
-Single stranded DNA (12 bp)
-Cohesive/sticky and complementary
ends
-DNA ligase (replication, repair and
recombination)
Linear
replication
Circular
© John Wiley & Sons, Inc.
Replication is Bidirectional
1-AT- and CG -rich regions
Native 100°C Denature
pH~11
~10 min
2-Bubbles
3-DNA polymerase access
© John Wiley & Sons, Inc.
Replication is Bidirectional
Both branch points ( Y
shape) are replication
forks
Replication fork: junction where the double-stranded DNA splits apart
(or unzipped) into 2 single strands.
© John Wiley & Sons, Inc.
Bidirectional Replication of Phage T7
-Phage T7
--eye structure
replication forks
© John Wiley & Sons, Inc.
• DNA replicates by a semiconservative mechanism:
as the two complementary strands of a parental
double helix unwind and separate, each serves as a
template for the synthesis of a new complementary
strand.
• The hydrogen-bonding potentials of the bases in
the template strands specify complementary base
sequences in the nascent DNA strands.
• Replication is initiated at unique origins and usually
proceeds bidirectionally from each origin.
© John Wiley & Sons, Inc.
DNA Replication in
Prokaryotes
DNA replication is a complex
process, requiring the concerted
action of a large number of proteins
© John Wiley & Sons, Inc.
DNA Polymerases and DNA Synthesis In Vitro
Much of what we know about DNA synthesis
was deduced from in vitro studies.
DNA Polymerase I
Single polypeptides
5’ to 3’
Triphosphate [dATP]
MgCl2
Free 3’OH group of the
DNA strands
© John Wiley & Sons, Inc.
Continuous vs discontinuous--leading
and lagging strands
Replicating fork
Bacteriophage T4
© John Wiley & Sons, Inc.
1,000 to 2,000 bp
Okazaki fragments
10,000 to 200,000 bp
Small fragments
to
© John Wiley & Sons, Inc.
big fragments
Continuous vs discontinuous--leading
and lagging strands
Replicating fork
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
Prepriming at oriC in E. coli
--Replication bubbles
Self aggregation
DNA helicase:
it separates two annealed nucleic acid
Strands.
Why?
© John Wiley & Sons, Inc.
RNA Primers are Used to Initiate DNA Synthesis
DNA primase: short RNA primer
RNA/DNA hybrid
(unstable ?)
Perfect conditions for
DNA polymerases to work
(free 3’OH)
© John Wiley & Sons, Inc.
DNA Polymerase I:
5'3' Polymerase Activity
© John Wiley & Sons, Inc.
DNA Polymerase I:
5'3' Exonuclease Activity
© John Wiley & Sons, Inc.
DNA Polymerase I:
3'5' Exonuclease Activity
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
DNA Helicase Unwinds the Parental Double Helix
One of the most important event during DNA replication
Why?
© John Wiley & Sons, Inc.
Single-Strand DNA Binding (SSB) Protein
Access to DNA polymerase
© John Wiley & Sons, Inc.
Supercoiling of Unwound DNA
DNA Topoisomerases I: produce single transient breaks of
DNA and remove supercoiling
It blocks DNA replication
DNA Topoisomerases II: produce double transient breaks of
DNA and negative supercoiling (DNA gyrase)
© John Wiley & Sons, Inc.
DNA
Topoisomerase I
Produces SingleStrand Breaks in
DNA
© John Wiley & Sons, Inc.
Requirements of DNA Polymerases
 Primer DNA with free 3'-OH
 Template DNA to specify the
sequence of the new strand
 Substrates: dNTPs
 Mg2+
 Reaction: nucleophilic attack
© John Wiley & Sons, Inc.
DNA Polymerase III is the True DNA Replicase of E. coli
DNA Polymerase III:
--a 900 KDa multimeric protein
--Dimers
--Holoenzymes
--High fidelity (error ~1 in a 1 x 1012)
© John Wiley & Sons, Inc.
Proofreading mechanism
 Subunits----Prokaryotes
 Subunits----Eukaryotes
© John Wiley & Sons, Inc.
The Replication Apparatus in E. coli
Primosome:
Initiation of Okazaki fragment during lagging strand
DNA primase and DNA helicase
DnaB and C proteins
Require ATP
DNA helicase:unwinds DNA
DNA primase: synthesis of RNA
Topoisomerase: transient DNA breaks
DNA polymerase III: extend the RNA primers
(deoyxribonucleotide). It is holoenzymes
© John Wiley & Sons, Inc.
DNA Replication
Synthesis of the leading strand is
continuous.
Synthesis of the lagging strand is
discontinuous. The new DNA is
synthesized in short segments (Okazaki
fragment) that are later joined together.
© John Wiley & Sons, Inc.
The E. coli Replisome
Replisome:
complete replication apparatus
© John Wiley & Sons, Inc.
Rolling-Circle Replication
Replication’s ModelsO-shape
Eye-shape
Y-shape
Rolling-circle (viruses, bacteria , amphibians)
_______________________________________
1- Nick by specific endonucleases
2-parental DNA is intact and functions as
template
3-DNA polymerase 5’ to 3’
4- displacement of one of the DNA strand
© John Wiley & Sons, Inc.
• DNA replication is complex, requiring the
participation of a large number of proteins.
• DNA synthesis is continuous on the progeny
strand that is being extended in the overall
5'3' direction, but is discontinuous on the
strand growing in the overall 3'5' direction.
© John Wiley & Sons, Inc.
• New DNA chains are initiated by short RNA
primers synthesized by DNA primase.
• DNA synthesis is catalyzed by enzymes
called DNA polymerases.
• All DNA polymerases require a primer
strand, which is extended, and a template
strand, which is copied.
© John Wiley & Sons, Inc.
• All DNA polymerases have an absolute
requirement for a free 3’-OH on the
primer strand, and all DNA synthesis
occurs in the 5’ to 3’ direction.
• The 3’ to 5’ exonuclease activities of
DNA polymerases proofread nascent
strands as they are synthesized,
removing any mispaired (match)
nucleotides at the 3’ termini of primer
strands.
© John Wiley & Sons, Inc.
• The enzymes and DNA-binding
proteins involved in replication
assembled into a replisome at each
replication fork and act in concert as the
fork moves along the parental DNA
molecule.
© John Wiley & Sons, Inc.
Unique Aspects of Eukaryotic
Chromosome Replication
Although the main features of
DNA replication are the same
in all organisms, some
processes occur only in
eukaryotes.
Viruses and E.coli
© John Wiley & Sons, Inc.
DNA Replication in Eukaryotes
Shorter RNA primers and Okazaki fragments
DNA replication only during S phase
(bacteria will duplicate DNA only in a rich environment)
Multiple origins of replication
(bacteria shows one origins of replication)
Cell Cycle
Nucleosomes
--check points
----S phase
----Mitosis
(nucleosomes are not present in bacteria)
Telomeres
(telomeres are not present in bacteria)
© John Wiley & Sons, Inc.
Bidirectional Replication from
Multiple Origins in Eukaryotes
Pulse chase experiments with 3H-thymidine
---Origins of replication
---Large number of replicons
(1 vs ~1x105)
© John Wiley & Sons, Inc.
Replicon:
segment of DNA
containing one
Origin (O) and
Two termini (T)
© John Wiley & Sons, Inc.
The Eukaryotic Replisome
SV40 virus: DNA virus (histones)
Bacteria replication
--unwind parental DNA (without histones)
----DNA helicase
----Topoisomerase
----Single -strand DNA binding protein
----DNA polymerase III
© John Wiley & Sons, Inc.
Eukaryotic Replication Proteins
Eukaryotic replication
----parental DNA (with histones)
Produce the
RNA/DNA chain
----Polymerases ()
-------Pol initiation of replication (origins)
priming of Okazaki fragment
complex with DNA primase
-------Pol synthesis of lagging strand
 Pol synthesis of leading strand
----accessories proteins: PCNA and Rf-C
(sliding clamp)
----Pol  have exonuclease activity ( 3’to 5”)
=proofreading
----Other Pols (pie, lambda, phi, rho, and mu)
do not have exonuclease activity ( 5’to 3”)
----Ribonulceases H1 and FEN-1
© John Wiley & Sons, Inc.
Proliferating Cell Nuclear Antigen: PCNA
Nucleosome Spacing in Replicating Chromatin
Assembly and disassembly of nucleosomes
© John Wiley & Sons, Inc.
Chromatin can have alternative states
Inactive--DNA/histones
Polymaerase/TFs
NO TRANSCRIPTION
Active--Polymerase/TFs
HISTONES
TRANSCRIPTION
“The addition of either TFs or nucleosomes may form stable structures
that can not be changed by modifying the equilibrium with free
components”
How is the chromatin structure regulated?
Chromatin remodeling
© John Wiley & Sons, Inc.
The Telomere “extension”
Problem
DNA polymerase can not replicate the terminal DNA
---too big
---not enough space ( 3’-OH, primer)
© John Wiley & Sons, Inc.
Telomerase
(Reverse Transcriptase)
G-rich telomere sequence
5’ to 3’
Aging (early aging….progerias)
Immortality:
Cancer and Normal cells
Senescence:
normal diploid cells cease to divide,
(about 45 to 50 cell divisions).
© John Wiley & Sons, Inc.
Telomere Length and Aging
 Shorter telomeres are
associated with cellular
senescence and death.
 Diseases causing premature
aging are associated with
short telomeres.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Apoptosis (self-destruction
):
© John Wiley & Sons, Inc.
programmed cell death
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this pic ture.
Telomeres Are Essential for
Survival
Figure 28.32
Dna polymerases classification as follows:
Prokaryotic DNA polymerases
Pol I to V
Eukaryotic DNA polymerases
Pol  theta, pie, lambda, phi, rho,
and mu.
Based on sequence homology
A, B, C, D, X, Y, and RT

bacterial
© John Wiley & Sons, Inc.
Since the parental double helix must rotate 360° to unwind each gyre of the helix,
during the semi-conservative replication of the bacterial chromosome, some kind of “swivel” must exist.
What do geneticists now know that the required swivel is?
a) Topoisomerase
b) Helicase
c) A transient single-strand break produced by the action of topoisomerases
d) A transient single-strand break produced by the action of helicases
e) A transient single-strand break produced by the action of Ligase
In the E. coli chromosome the origin of replication, called oriC, is characterized as being rich in:
a) A-G base pairs
b) A-C base pairs
c) C-G base pairs
d) C-T base pairs
e) None of the above
Telomere length has not been correlated with:
a) Aging
b) Sex determination
c) Progeria
d) Cancer
e) All of these