DNA replication_NR_2011(1)

8/7/2019 DNA replication_NR_2011(1)

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DNA Replication


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S phase

G2phase

M

phase

G1

phase

DNA & Histone

synthesis

Eukaryotic Cell CyclesEukaryotic Cell Cycles

Mitosis (M)

takes placeonly after DNA

synthesis (S).

Two gaps (G1

and G2) intime separate

the two

processes.


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DNA Replication

???????????


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Duplication of DNA

New DNA strands are synthesized by using the existing

(parental) strands as templates in the formation of new,daughter strands complementary to the parental strands

DNA Replication

High degree of fidelity

Both the strands of parental DNA serve as templates and

synthesis occurs simultaneously on both the strands

DNA replication is bidirectional

Always 5 3 (template is read in????)


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DNA Replication

Each of the parental

strands serves as a

template for a daughter

strand

Semi conservative


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DNA Replication

Requirements:Templates

Deoxy ribonucleotides (dATP, dGTP,dCTP,TTP)

Enzymes to polymerize

Unwinding/separation of the strandsMaintenance of single stranded status

Primer

Editing (Proof Reading)


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Prokaryotic DNA Replication

1. Origin of replication (oriC locus)


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1. Origin of replication (oriC locus)

a. Rich in AT base pairs ???

b. In E Coli, the oriC is bound by the protein dnawhich brings local denaturation and unwinding of

adjacent AT region of DNA (melting)

c. ATP requiring process


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2. Unwinding of DNA

a.The interaction of proteins with ori defines the start site

of replication and provides a short region of ss DNA

essential for initiation of synthesis the nascent DNA

strandb. Requires the formation of a number of protein-protein

and protein-DNA interaction

c. DNA helicase using ATP hydrolysis allows progressive

unwinding of DNA. In E.coli it is a complex ofdnaB helicase and dnaC protein

d. Maintainence of unwinding state and free from

nucleases attack--

Single-stranded DNA binding proteins (SSBs)


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2. Unwinding of DNA (contd..)

DNA + Helicase complex + SSBs = prepriming complex


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3. Formation of replication fork

Has 4 components that form in the following sequence


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3. Formation of replication fork (contd..)

i. The DNA helicase unwinds a short segment of the

parental duplex DNA

ii. A primase initiates synthesis of an RNA moleculethat is essential for priming DNA synthesis and is

complementary to the unwound template strands?

(primer is about 10-200 ribonucleotides)

Primase + prepriming complex = Primosomes

(The mobile complex)


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Prokaryotic DNA Replication3. Formation of replication fork (contd..)

iii. The primer, still base-paired to its complementary

DNA strand, is then elongated by a DNA polymerasenew daughter strand


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3. Formation of replication fork (contd..)

iv. SSBs bind to a ssDNA and prevent premature

reannealing of ssDNA to form dsDNA


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DNA PolymerasesPolymerises deoxyribonucleotides

Synthesis DNA in the 5 3

(No polymerase in 3 5)

Template-directed enzymes

Needs RNA primerDifferent types of DNA polymerases

Share important 3 properties

1. Chain Elongation

2. Processivity (an expression of the # of nucleotide added to the

nascent chain before the polymerase disengagesfrom the template)

2 identical subunit of Pol III forms a clamp "around the

template

3. Proof Reading (Identifies copying errors and corrects them)


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Functions of DNA polymerases

DNA polymerases of E. coli

pol I pol II pol III (core)

Polymerization: 5 to 3 yes yes yesProofreading exonuclease: 3 to 5 yes yes yes

Repair exonuclease: 5 to 3 yes no no

DNA polymerase III is the main replicating enzymeDNA polymerase I has a role in replication to fill gaps and excise

primers on the lagging strand, and it is also a repair enzyme

and is used in making recombinant DNA molecules

DNA polymerase II participates in DNA repair



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Major Steps In Replication

1. Initiation

2. Elongation

3.Termination

INITIATION

1. Origin of replication (oriC): dnaA2. Unwinding of DNA: Helicases- dnaB and dnaC

SSB

3. Synthesis of RNA primer: Primase(formation of DNA-RNA hybrid)

4. Entry of first dNTP:DNA polymerase III


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Prokaryotic DNA ReplicationELONGATION

1. DNA Polymerase III (5 3 polymerase activity)2. The template dictates which dNTP is complementary and by

hydrogen bonding holds it in a place while the 3 OH group

of the growing strand attacks and incorporates the new

nucleotide into the polymer(For picture see the slide #21)

3. subunit of Pol III forming a clamp "around the template4. Only a small stretch of the template duplex is single-

stranded at any given time.

5. Formation of replication fork and leading and lagging

strand.(Semi discontinuous DNA synthesis)

6. Formation and deformation of super coils (for details ref slide #22-24)

7. Proof Reading: DNA Polymerase III (3 5 exonuclease

activity)

8. Bidirectional (For picture see the slide #25)

9. Reformation of the helix


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Initiation

Elongation


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ELONGATION

(contd)The progression of fork requires continuous unwinding of DNA

Severe torsional stress into the duplex ahead of the fork

DNA Topoisomerase

1. Cleaves 1 (Type I) or both (Type II) strands of DNA

(transient nick)2. Unwinding of broken end around the intact strand

3. Resealing


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ELONGATION

(contd)2 Types of topoisomerasesType I

- Cleaves 1 strand

- No ATP involvement

Type II- Cleaves both the strands

- Needs ATP

eg: DNA gyrase

- Bacteria and plants

- Neutralises positive super coils by introducingnegative super coils

Both the types are important in replication, transcription and

recombination


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ELONGATION

(contd)


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TERM

INATION(part of EL

ONGATION??)

?????????


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TERM

INATION(contd)1. Removal of primers: Pol I (5 3 exonuclease activity)

2. Replacement of them by DNA: Pol I (5 3 polymerase activity)

3. Proof Reading: Pol I (3 5 exonuclease activity)

4. Ligation: DNA Ligase

The final phosphodiester linkage between 5 phosphate group

synthesized by Pol III and 3 OH group made by Pol I is catalyse

by DNA ligase and is ATP-dependent

The bacterial DNA molecule circularizes at the end of the

DNA ligase


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Eukaryotic DNA Replication

Essentially similar to prokaryotic replication- Bidirectional

- RNA primers

- Leading and lagging strand

Bacterial genome is about 6X106 bpReplication is completed in ~ 30 min

Replication rate is ~ 3X105 bp/min

Eukaryotic genome is about 3X109 bp

If the replication rate is 3X105 bp/min from a single Ori, then replicationwould be for over 150 hrs!!!!

Solution????


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1. Bidirectional

2. Multiple origins in each chromosome

generates Replication Bubbles


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PolymerasePolymerase FunctionFunction Proof readingProof reading

ContainsContains

primaseprimase;initiates;initiates

replicationreplication

--

Repair Repair --

ReplicatesReplicates

mitochondrial DNAmitochondrial DNA

++

ElongatesElongates ++

Repair Repair ++

Eukaryotic DNA polymerases


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DNA polymerase = RNA polymerase (primase)!!!!!!!!

+

DNA polymerase

DNA polymerase = Elongation

Topoisomerase I (check prokaryotic part) (is it a DNA ligase?

and or endonuclease?


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PCNA (Proliferating Cell NuclearAntigen)

- Acts like subunit of DNA polymerase III ????

- Binds to DNA polymerase

- PCNA method is used as Mitotic Index


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Telomeres and Telomerase


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Telomer is a region of repetitive DNA sequence at the end of aChromosome (AGGGTT)


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- Chromosome/DNA can not replicate right to the tip-Limited somatic cell division

Sequences are lost in every replicative phase

cell division stops

Ageing!!!!!!! (senescence)

If sequences are protected from loss

Immortal !!!

Critical level

Stem cells, germ

cells, Cancer cells


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5 3

Primer on lagging strand

5

3

-No Polymerase to fill the gap

-One long and one short strand

-Protect the single strand???

-Continues, reaches a critical level-Cell ages !!

Solution in stem cells, germ cells???


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Loss is inevitable

Loss is replenished!!!

5 3

3 5

5

3

Next replication


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Telomerase is a unique reverse transcriptase

- A cellular reverse transcriptase

- Contains intrinsic RNA component (ribonucleoprotein)- Specializes in synthesizing multiple repeats


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Reverse Transcriptase

Retroviruses (HIV) genome is ss RNA

After infection of host cell, viral reverse transcriptase

using the RNA as template synthesises DNA reversetranscription

Lacksproofreading activity high mutation rate

responsible for the development of drug resistance and

difficulties in developing a vaccine


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Chemotherapies Targeting Replication

Alkylating agents Ex: cyclophosphamide, chlorambucilfunction by

cross-linking of bases in the DNA

induce mispairing of nucleotides act upon DNA at all stages of the cell cycle

Anthracyclines Ex : Doxorubicin inhibit the actions of topoisomerase II

Etoposide inhibition of topoisomerase II Camptothecins inhibit the action of topoisomerase I anticancer drugs that interfere with nucleotide metabolism

antimetabolites

Any other ???????????

DNA replication_NR_2011(1)

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