Replication of DNA

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Outline
Basic Features of DNA Replication In Vivo

DNA Replication in Prokaryotes

Unique Aspects of Eukaryotic DNA
Replication
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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

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DNA Replication is Semiconservative
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
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MODEL
Possible Models of
DNA Replication
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The Meselson-Stahl Experiment:
DNA Replication in E. coli is Semiconservative
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Bacteria growing with
15
N for several generations



Change medium and add
14
N

--one generation
--two generations
--three generations




The Origin of Replication in E. coli
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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
Mer=repeating unit=parts
Eukaryotic:
ARS
(Autonomously Replicating sequences)
AT-rich region 11bp
N: any nucleotide
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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.
Bidirectional Replication: The Phage Chromosome
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-Small bacterial virus

-Single stranded DNA (12 bp)

-Cohesive/sticky and complementary
ends

-DNA ligase (replication, repair and
recombination)

Linear

Circular
replication
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.
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DNA Replication in
Prokaryotes
DNA replication is a complex
process, requiring the concerted
action of a large number of proteins
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DNA Polymerases and DNA Synthesis In Vitro
Much of what we know about DNA synthesis
was deduced from in vitro studies.
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DNA Polymerase I
Single polypeptides

5 to 3

Triphosphate [dATP]

MgCl
2

Free 3OH group of the DNA strands
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Continuous vs discontinuous--leading
and lagging strands
Replicating fork
Bacteriophage T4
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Continuous vs discontinuous--leading
and lagging strands
Replicating fork
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Prepriming at oriC in E. coli
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--Replication bubbles
Self aggregation
Why?
DNA helicase:
it separates two annealed nucleic acid
Strands.
RNA Primers are Used to Initiate DNA Synthesis
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DNA primase: short RNA primer
RNA/DNA hybrid
(unstable ?)

Perfect conditions for
DNA polymerases to work
(free 3OH)
DNA Polymerase I:
5'3' Polymerase Activity
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DNA Polymerase I:
5'3' Exonuclease Activity
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DNA Polymerase I:
3'5' Exonuclease Activity
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DNA Helicase Unwinds the Parental Double Helix
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One of the most important event during DNA replication
Why?
Single-Strand DNA Binding (SSB) Protein
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Access to DNA polymerase
Supercoiling of Unwound DNA
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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)
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DNA Topoisomerase I
Produces Single Strand
Breaks in DNA
Requirements of DNA Polymerases
Primer DNA with free 3'-OH

Template DNA to specify the
sequence of the new strand

Substrates: dNTPs

Mg
2+


Reaction: nucleophilic attack
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DNA Polymerase III is the True DNA Replicase of E. coli
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DNA Polymerase III:
--a 900 KDa multimeric protein
--Dimers
--Holoenzymes









--High fidelity (error ~1 in a 1 x 10
12
)
Proofreading mechanism
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c Subunits----Prokaryotes

co Subunits----Eukaryotes

The Replication Apparatus in E. coli
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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



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.
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The E. coli Replisome
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Replisome:
complete replication apparatus

Rolling-Circle Replication
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Replications Models-
O-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
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.
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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.

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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.
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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.
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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.
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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)

Nucleosomes
(nucleosomes are not present in bacteria)

Telomeres
(telomeres are not present in bacteria)
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Cell Cycle

--check points
----S phase
----Mitosis
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Replicon:
segment of DNA
containing one
Origin (O) and
Two termini (T)

The Eukaryotic Replisome
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SV40 virus: DNA virus (histones)
Bacteria replication
--unwind parental DNA (without histones)
----DNA helicase
----Topoisomerase
----Single -strand DNA binding protein
----DNA polymerase III
Eukaryotic Replication Proteins
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Eukaryotic replication
----parental DNA (with histones)

----Polymerases (o,o,c)

-------Pol o: initiation of replication (origins)
priming of Okazaki fragment
complex with DNA primase

-------Pol o: synthesis of lagging strand

Pol c: synthesis of leading strand
----accessories proteins: PCNA and Rf-C
(sliding clamp)

----Pol o,, c have exonuclease activity ( 3to 5)
=proofreading

----Other Pols (pie, lambda, phi, rho, and mu)
do not have exonuclease activity ( 5to 3)

----Ribonulceases H1 and FEN-1
Produce the
RNA/DNA chain
Proliferating Cell Nuclear Antigen: PCNA
Nucleosome Spacing in Replicating Chromatin
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Assembly and disassembly of nucleosomes
Chromatin can have alternative states
Inactive--DNA/histones





Active--Polymerase/TFs
Polymaerase/TFs
NO TRANSCRIPTION
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?
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Chromatin remodeling
The Telomere extension
Problem
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DNA polymerase can not replicate the terminal DNA
---too big
---not enough space ( 3-OH, primer)
Telomerase
(Reverse Transcriptase)

G-rich telomere sequence
5 to 3

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Aging (early aging.progerias)

Immortality:
Cancer and Normal cells

Senescence:
normal diploid cells cease to divide,
(about 45 to 50 cell divisions).

Telomere Length and Aging

Shorter telomeres are
associated with cellular
senescence and death.
Diseases causing premature
aging are associated with
short telomeres.
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Apoptosis (self-destruction):
programmed cell death
Telomeres Are Essential for
Survival
Figure 28.32
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Dna polymerases classification as follows:

Prokaryotic DNA polymerases
Pol I to V

Eukaryotic DNA polymerases
Pol o,|,o,c, theta, pie, lambda, phi, rho,
and mu.

Based on sequence homology
A, B, C, D, X, Y, and RT
o,o,c
bacterial
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