chapter 10 replication of dna © john wiley & sons, inc

Chapter 10Replication 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


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


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


MODEL

Possible Models ofDNA Replication


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

Centrifugation:process involving the centrifugal force for the sedimentation of particles and/or molecules

[revolutions per minute (RPM)]

gravitational force to cause precipitation/sedimentation

Bacteria with 15N-parental(several periods of time)

Bacteria with 14N-daugther

http://en.wikipedia.org/wiki/Centrifugal_force

http://en.wikipedia.org/wiki/Revolutions_per_minute



http://en.wikipedia.org/wiki/Gravitation

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

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


3H-Thymidine

1H-Thymidine

C-metaphase: Colchicine-metaphase:Colchincine: is a toxic natural product and secondary metabolite and it inhibits microtubule polymerization by binding to tubulin.

http://en.wikipedia.org/wiki/Natural_product

http://en.wikipedia.org/wiki/Secondary_metabolite

http://en.wikipedia.org/wiki/Microtubule

http://en.wikipedia.org/wiki/Tubulin

http://en.wikipedia.org/wiki/Tubulin

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

Mer=repeating unit=parts

Eukaryotic: ARS(Autonomously Replicating sequences)AT-rich region 11bp

N: any nucleotide


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.

Visualization of Replication in E. coli


3H-Thymidine

Autoradiography

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

Circular

replication

Replication is Bidirectional


1-AT- and CG -rich regions

Native Denature

2-Bubbles

3-DNA polymerase access

100°CpH~11~10 min

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.

Bidirectional Replication of Phage T7


-Phage T7

--eye structure

replication forks

• 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.


DNA Replication in Prokaryotes

DNA replication is a complex process, requiring the concerted action of a large number of proteins


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


Continuous vs discontinuous--leading and lagging strands

Replicating fork

Bacteriophage T4


1,000 to 2,000 bp

10,000 to 200,000 bp

Small fragments to big fragments

Okazaki fragments


Continuous vs discontinuous--leading and lagging strands

Replicating fork

Prepriming at oriC in E. coli


--Replication bubbles

Self aggregation

Why?

DNA helicase: it separates two annealed nucleic acid Strands.

http://en.wikipedia.org/wiki/Nucleic_acid_thermodynamics#Annealing

RNA Primers are Used to Initiate DNA Synthesis


DNA primase: short RNA primerRNA/DNA hybrid(unstable ?)

Perfect conditions for DNA polymerases to work(free 3’OH)

DNA Polymerase I:5'3' Polymerase Activity


DNA Polymerase I:5'3' Exonuclease Activity


DNA Polymerase I:3'5' Exonuclease Activity


DNA Helicase Unwinds the Parental Double Helix


One of the most important event during DNA replication

Why?

Single-Strand DNA Binding (SSB) Protein


Access to DNA polymerase

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)

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

Mg2+

Reaction: nucleophilic attack


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)

Proofreading mechanism


Subunits----Prokaryotes

Subunits----Eukaryotes

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

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.


The E. coli Replisome


Replisome: complete replication apparatus

Rolling-Circle Replication


Replication’s 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.


• 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.


• 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.


• 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.


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

DNA Replication in EukaryotesShorter 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)


Cell Cycle

--check points----S phase----Mitosis

Bidirectional Replication from Multiple Origins in Eukaryotes


Pulse chase experiments with 3H-thymidine

---Origins of replication

---Large number of replicons

(1 vs ~1x105)


Replicon: segment of DNAcontaining one Origin (O) andTwo termini (T)

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

Eukaryotic Replication Proteins


Eukaryotic replication----parental DNA (with histones)

----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

Produce the RNA/DNA chain

Proliferating Cell Nuclear Antigen: PCNA

Nucleosome Spacing in Replicating Chromatin


Assembly and disassembly of nucleosomes

Chromatin can have alternative states

Inactive--DNA/histones

Active--Polymerase/TFs

Polymaerase/TFsNO 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?


Chromatin remodeling

The Telomere “extension” Problem


DNA polymerase can not replicate the terminal DNA---too big ---not enough space ( 3’-OH, primer)

Telomerase(Reverse Transcriptase)

G-rich telomere sequence5’ to 3’


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


Dna polymerases classification as follows:

Prokaryotic DNA polymerasesPol 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

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) Topoisomeraseb) Helicasec) A transient single-strand break produced by the action of topoisomerasesd) A transient single-strand break produced by the action of helicasese) 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 pairsb) A-C base pairsc) C-G base pairsd) C-T base pairse) None of the above

Telomere length has not been correlated with:

a) Agingb) Sex determinationc) Progeriad) Cancere) All of these

chapter 10 replication of dna © john wiley & sons, inc

Documents

coli john wiley sons

nucleotides john wiley

generations john wiley

tritium john wiley sons

nucleotide john wiley

model john wiley sons

eukaryotes john wiley

ndaugther john wiley