DNA Model Building and Replication

DNA Replication – S phase

Origins of replication in E. coli and eukaryotesOrigin of replication

0.5

µm

0.25

µm

Bacterialchromosome

Two daughterDNA molecules

Replicationbubble

Parental (template)strand Daughter

(new) strand

Replicationfork

Double-strandedDNA molecule

(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cellOrigin ofreplication Eukaryotic chromosome

Double-strandedDNA molecule

Parental (template)strand

Daughter (new) strand

Replicationfork

Bubble

Two daughter DNA molecules

Wow! Hundreds or even thousands of origins of replication in eukaryotes.

Only one originof replication in prokaryotes.

The Trombone Model – DNA replication resembles the slide of a trombone

Leading strand template

5ʹ

5ʹ

5ʹ

5ʹ 3ʹ

3ʹ

3ʹ

3ʹ

3ʹ 3ʹ

5ʹ

5ʹ

Leading strand

Lagging strandLagging strandtemplate

DNA pol IIIConnecting protein

Helicase

Parental DNA

DNA pol III

“DNA Replication Machine” proteins involved in the initiation of DNA replication

Topoisomerase

Primase

RNAprimer

Replicationfork

5ʹ3ʹ

5ʹ

5ʹ

3ʹ

HelicaseSingle-strand bindingproteins

3ʹ

• At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating.

• Helicases are enzymes that untwist the double helix at the replication forks.

• Single-strand binding proteins bind to and stabilize single-stranded DNA.

• Topoisomerase corrects for �overwinding� ahead of replication forks by breaking, swiveling, and rejoining DNA strands. Preparation for replication of DNA; breaks the phosphate backbone of the DNA helix.

“DNA Replication Machine” proteins involved in the initiation of DNA replication

Topoisomerase

Primase

RNAprimer

Replicationfork

5ʹ3ʹ

5ʹ

5ʹ

3ʹ

HelicaseSingle-strand bindingproteins

3ʹ

5ʹ 5ʹ3ʹ

3ʹ

5ʹ 5ʹ

New strand Template strand

Sugar

Phosphate Base

OH

A T

C

C

G

G

AT

COH

Nucleotide

DNApoly-

merase

OH

Pyro-phosphate

2 P i

P iPP PP

3ʹ

3ʹ

A T

C

C

G

G

A

C

T

Incorporation of a nucleotide into a DNA strand

• DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to an existing 3ʹ end.

• The initial nucleotide strand is a short RNA primer.

Topoisomerase

Primase

RNAprimer

Replicationfork

5ʹ

3ʹ

5ʹ

5ʹ

3ʹ

HelicaseSingle-strand bindingproteins

3ʹ

• An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template.

• The primer is short (5–10 nucleotides long), and the 3ʹ end serves as the starting point for the new DNA strand.

Topoisomerase

Primase

RNAprimer

Replicationfork

5ʹ3ʹ

5ʹ

5ʹ

3ʹ

HelicaseSingle-strand bindingproteins

3ʹ

Synthesizing a New DNA Strand

• Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork.

• Most DNA polymerases require a primer and a DNA template strand.

• The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells.

5ʹ 5ʹ3ʹ

3ʹ

5ʹ 5ʹ

New strand Template strand

Sugar

Phosphate Base

OH

A T

C

C

G

G

AT

COH

Nucleotide

DNApoly-

merase

OH

Pyro-phosphate

2 P i

P iPP PP

3ʹ

3ʹ

A T

C

C

G

G

A

C

T

Incorporation of a nucleotide into a DNA strand

Antiparallel Elongation• The antiparallel structure of the double helix affects

replication.• DNA polymerases add nucleotides only to the free 3ʹ end

of the growing strand; therefore, a new DNA strand can elongate only in the 5ʹ to 3ʹ direction.

Leading strand Lagging strand

Leading strandLagging strand

Primer

Origin of replication

Overall directionsof replication

3’5’

5’3’

Synthesis of the leading strand during DNA replication

Leading strand Lagging strand

Leading strand

Parental DNA

Lagging strand

Primer

Origin of replication

Overalldirections

of replication Origin of replication

RNA primerSliding clamp

DNA pol III

Continuouselongation in the5ʹ to 3ʹ direction

5ʹ

5ʹ

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ 3ʹ

3ʹ3ʹ

3ʹ

3ʹDNA pol III starts tosynthesize leadingstrand.

1

2

Leading strand Lagging strand

Leading strandLagging strand

Primer

Overall directionsof replication

3’5’

5’3’

Origin of replication

Synthesis of the leading strand during DNA replication

Parental DNA

Origin of replication

RNA primerSliding clamp

DNA pol III

5ʹ

5ʹ

5ʹ

3ʹ 3ʹ

3ʹ

DNA pol III starts tosynthesize leadingstrand.

1

5ʹ3ʹ

5ʹ3ʹ

5ʹ

3ʹ

2 Continuouselongation in the5ʹ to 3ʹ direction

• Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork.

Leading strand Lagging strand

Leading strandLagging strand

Primer

Overall directionsof replication

3’5’

5’3’

Origin of replication

• To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork

• The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase.

Synthesis of the lagging strand

Overview

12

1

1

21

21

1

2

5ʹ

3ʹ

3ʹ

5ʹ5ʹ3ʹ3ʹ

3ʹ 3ʹ5ʹ

5ʹ5ʹ

5ʹ3ʹ

3ʹ

5ʹ3ʹ

5ʹ3ʹ

5ʹ3ʹ

5ʹ3ʹ

5ʹ3ʹ

5ʹ3ʹ

1

2 5

6

4

3

Origin of replicationLaggingstrand

Leadingstrand

Laggingstrand

LeadingstrandOverall directions

of replication

RNA primerfor fragment 2

Okazakifragment 2

DNA pol IIImakes Okazakifragment 2.

DNA pol Ireplaces RNAwith DNA.

DNA ligaseforms bondsbetween DNAfragments.

Overall direction of replication

Okazakifragment 1

DNA pol IIIdetaches.

RNA primerfor fragment 1

Templatestrand

DNA pol IIImakes Okazakifragment 1.

Origin ofreplication

Primase makesRNA primer.

5ʹ

Figure 16.16a

12

OverviewOrigin of replication

Laggingstrand

Leadingstrand

Laggingstrand

Leadingstrand

Overall directionsof replication

Synthesis of the lagging strand

Figure 16.16b-1

5ʹ

3ʹ

5ʹ5ʹ3ʹ3ʹ

Templatestrand

Origin ofreplication

Primase makesRNA primer.

1

Synthesis of the lagging strand(Step 1)

Figure 16.16b-2

5ʹ

3ʹ

5ʹ5ʹ3ʹ3ʹ

Templatestrand

Origin ofreplication

Primase makesRNA primer.

1

5ʹ3ʹ

5ʹ

3ʹ

5ʹ3ʹ

RNA primerfor fragment 1

DNA pol IIImakes Okazakifragment 1.

1

2

Synthesis of the lagging strand(Step 2)

5ʹ

3ʹ

5ʹ5ʹ3ʹ3ʹ

Templatestrand

Origin ofreplication

Primase makesRNA primer.

1

5ʹ3ʹ

5ʹ

3ʹ

5ʹ3ʹ

RNA primerfor fragment 1

DNA pol IIImakes Okazakifragment 1.

1

2

1

33ʹ

3ʹ5ʹ

5ʹOkazakifragment 1

DNA pol IIIdetaches.

Synthesis of the lagging strand(Step 3)

DNA pol IIImakes Okazakifragment 2.2

5ʹ3ʹ

5ʹ3ʹ

4

RNA primerfor fragment 2

Okazakifragment 2

1

Synthesis of the lagging strand(Step 4)

DNA pol IIImakes Okazakifragment 2.2

5ʹ3ʹ

5ʹ3ʹ

4

RNA primerfor fragment 2

Okazakifragment 2

1

5ʹ3ʹ

DNA pol Ireplaces RNAwith DNA.

5ʹ3ʹ

12

5

Synthesis of the lagging strand(Step 5)

DNA ligaseforms bondsbetween DNAfragments.

Overall direction of replication

DNA pol IIImakes Okazakifragment 2.2

5ʹ3ʹ

5ʹ3ʹ

4

RNA primerfor fragment 2

Okazakifragment 2

1

5ʹ3ʹ

DNA pol Ireplaces RNAwith DNA.

5ʹ3ʹ

12

5

6

5ʹ3ʹ

5ʹ3ʹ

12

Synthesis of the lagging strand(Step 6)

The Trombone Model – DNA replication resembles the slide of a trombone

Leading strand template

5ʹ

5ʹ

5ʹ

5ʹ 3ʹ

3ʹ

3ʹ

3ʹ

3ʹ 3ʹ

5ʹ

5ʹ

Leading strand

Lagging strandLagging strandtemplate

DNA pol IIIConnecting protein

Helicase

Parental DNA

DNA pol III

Summary of key concepts: DNA replicationDNA pol III synthesizesleading strand continuously

ParentalDNA

Helicase

Primase synthesizesa short RNA primer

3ʹ5ʹ

DNA pol I replaces the RNAprimer with DNA nucleotides

3ʹ5ʹ

3ʹ5ʹ

DNA pol III starts DNAsynthesis at 3ʹ end of primer,continues in 5ʹ → 3ʹ direction

Lagging strand synthesizedin short Okazaki fragments,later joined by DNA ligase

Origin of replication

Proofreading and Repairing DNA:• DNA polymerases proofread newly made DNA, replacing

any incorrect nucleotides.• In mismatch repair of DNA, repair enzymes correct errors

in base pairing.• DNA can be damaged by exposure to harmful chemical or

physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes.

• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA.

Nuclease

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ

5ʹ

5ʹ

3ʹ

3ʹ

DNApolymerase

DNAligase

5ʹ

3ʹ5ʹ

3ʹ

Nucleotide excision repair of DNA damage

Replicating the Ends of DNA Molecules• Limitations of DNA polymerase create problems for the

linear DNA of eukaryotic chromosomes• The usual replication machinery provides no way to

complete the 5ʹ ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends

• This is not a problem for prokaryotes, most of which have circular chromosomes.

• Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called telomeres.

• Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules.

• It has been proposed that the shortening of telomeres is connected to aging.

• If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce.

• An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells.

• The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions.

• There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist.

"Telomerase is crucial for telomere maintenance and genome integrity," explains Julian Chen, professor of chemistry and biochemistry at ASU and one of the project's senior authors. "Mutations that disrupt telomerase function have been linked to numerous human diseases that arise from telomerase gene activity.

The enzyme telomerase may help unlock secrets of aging and cancer

So, grab a “DNA Replication Machine Kit” and start duplicating!

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