Chapter 16 DNA Synthesis. Extra Credit – due by Jan. 24, 2014 Up to +24 points added to lowest...

Chapter 16Chapter 16DNA SynthesisDNA Synthesis

Extra Credit – due by Jan. 24, 2014Extra Credit – due by Jan. 24, 2014

Up to +24 points added to lowest quiz grade Research up to 6 bacterial diseases. For each:

1. State the name of the disease

2. State the bacterial Genus and species name, spelled correctly (such as Escherichia coli)

3. Describe the major symptoms of the disease

4. Describe how the disease is currently treated

Up to +24 points added to lowest quiz grade Research up to 6 bacterial diseases. For each:

1. State the name of the disease

2. State the bacterial Genus and species name, spelled correctly (such as Escherichia coli)

3. Describe the major symptoms of the disease

4. Describe how the disease is currently treated

Deoxyribonucleic AcidDeoxyribonucleic Acid

DNA, the substance of inheritance Is the most celebrated molecule of our time

Hereditary information Is encoded in the chemical language of DNA and

reproduced in all the cells of your body

DNA Contains the instructions for making proteins

DNA, the substance of inheritance Is the most celebrated molecule of our time

Hereditary information Is encoded in the chemical language of DNA and

reproduced in all the cells of your body

DNA Contains the instructions for making proteins

The Hershey – Chase experimentThe Hershey – Chase experiment

Bacteriophage with phosphorus-32 in DNA

Phage infectsbacterium

Radioactivity inside bacterium

Bacteriophage with sulfur-35 in protein coat

Phage infectsbacterium

No radioactivity inside bacterium

DNA, not protein, contains genes!http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html


Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Structure of DNADNA is a polymer of nucleotides

Each consisting of three components: a nitrogenous base, a sugar, and a phosphate group Sugar-phosphate

backboneNitrogenous

bases

5 endO–

O P O CH2

5

4O–

HH

OH

HH

3

1H O

CH3

N

O

NH

Thymine (T)

O

O P OO–

CH2

HH

OH

HH

HN

N

N

H

NH

H

Adenine (A)O

O P O

O–

CH2

HH

OH

HH

HH H

HN

NN

OCytosine (C)

O

O P O CH2

5

4O–

H

O

HH

3

1

OH2

H

N

NN H

ON

N HH

H H

Sugar (deoxyribose)3 end

Phosphate

Guanine (G)

DNA nucleotide

2

N


Structure of DNA Watson and Crick deduced that DNA was a

double helix

The double helix allowed for a mechanism of replication C

T

A

A

T

CG

GC

A

C G

AT

AT

A T

TA

C

TA0.34 nm

3.4 nm

(a) Key features of DNA structure

G

1 nm

G

(c) Space-filling model

T


Base Pairing RulesO

–O O

OH

O

–OO

O

H2C

O

–OO

O

H2C

O

–OO

O

OH

O

O

OT A

C

GC

A T

O

O

O

CH2

OO–

OO

CH2

CH2

CH2

5 end

Hydrogen bond3 end

3 end

G

P

P

P

P

O

OH

O–

OO

O

P

P

O–

OO

O

P

O–

OO

O

P

H2C

5 end

O


Overview of Semi-Conservative DNA Replication In DNA replication

The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

(a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

(b) The first step in replication is separation of the two DNA strands.

(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C


Replication Begins

A eukaryotic chromosome

May have hundreds or even thousands of replication origins

Replication begins at specific siteswhere the two parental strandsseparate and form replicationbubbles.

The bubbles expand laterally, asDNA replication proceeds in bothdirections.

Eventually, the replicationbubbles fuse, and synthesis ofthe daughter strands iscomplete.

1

2

3

Origin of replication

Bubble

Parental (template) strand

Daughter (new) strand

Replication fork

Two daughter DNA molecules

In eukaryotes, DNA replication begins at many sites along the giantDNA molecule of each chromosome.

In this micrograph, three replicationbubbles are visible along the DNA ofa cultured Chinese hamster cell (TEM).

(b)(a)

0.25 µm


New strand Template strand5 end 3 end

Sugar A TBase

C

G

G

C

A

C

T

PP

P

OH

P P

5 end 3 end

5 end 5 end

A T

C

G

G

C

A

C

T

3 end

2 P

OH

Phosphate

Elongating a New DNA Strand Elongation of new DNA at a replication fork

Is catalyzed by enzymes called DNA polymerases, which add nucleotides to the new daughter strand

Other Enzyme “Helpers”Other Enzyme “Helpers”

Helicase Unwinds the parental DNA strand to allow new

incoming nucleotides access

Single Stranded Binding Proteins Keep unwound strands apart during base

pairing

Helicase Unwinds the parental DNA strand to allow new

incoming nucleotides access

Single Stranded Binding Proteins Keep unwound strands apart during base

pairing

Limitations of DNA PolymeraseLimitations of DNA Polymerase

Limitation #1 DNA polymerase adds nucleotides

Only to the free 3end of a growing strand

Along one template strand of DNA, the leading strand, DNA polymerase can synthesize a complementary

strand continuously, moving toward the replication fork

Limitation #1 DNA polymerase adds nucleotides

Only to the free 3end of a growing strand

Along one template strand of DNA, the leading strand, DNA polymerase can synthesize a complementary

strand continuously, moving toward the replication fork

Limitations of DNA PolymeraseLimitations of DNA Polymerase To elongate the other new strand of DNA, 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 then joined together by DNA ligase

To elongate the other new strand of DNA, 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 then joined together by DNA ligase

Meselson – Stahl ExperimentMeselson – Stahl Experiment

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#








Synthesis of leading and lagging strands during DNA replication

Synthesis of Leading vs. Lagging Strand

Parental DNA

DNA pol elongatesDNA strands only in the5 3 direction.

1

Okazakifragments

DNA pol III

Templatestrand

Lagging strand3

2

Templatestrand DNA ligase

Overall direction of replication

One new strand, the leading strand,can elongate continuously 5 3 as the replication fork progresses.

2

The other new strand, thelagging strand must grow in an overall3 5 direction by addition of shortsegments, Okazaki fragments, that grow5 3 (numbered here in the orderthey were made).

3

DNA ligase joins Okazakifragments by forming a bond betweentheir free ends. This results in a continuous strand.

4

35

5

3

35

21

Leading strand

1


Lagging – strand synthesis is complex

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#



Limitations of DNA PolymeraseLimitations of DNA Polymerase

Limitation #2 DNA polymerase cannot initiate the synthesis

of a polynucleotide They can only add new nucleotides to existing

paired nucleotides

The initial nucleotide strand Is a RNA primer RNA primers are made with the help of the

enzyme primase

Limitation #2 DNA polymerase cannot initiate the synthesis

of a polynucleotide They can only add new nucleotides to existing

paired nucleotides

The initial nucleotide strand Is a RNA primer RNA primers are made with the help of the

enzyme primase


Priming of DNA SynthesisPrimase joins RNA nucleotides into a primer.

1

DNA pol adds DNA nucleotides to the primer, forming an Okazaki fragment.

2

After reaching the next RNA primer (not shown), DNA pol falls off.

3

After the second fragment is primed. DNA pol adds DNAnucleotides until it reaches the first primer and falls off.

4

DNA pol replaces the RNA with DNA, adding to the 3 end of fragment 2.

5

DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1.

6 The lagging strand in this region is nowcomplete.

7

Overall direction of replication

3

3

3

35

35

35

35

3

5

3

5

3

5

3 5

5

1

1

21

12

5

5

12

35

Templatestrand

RNA primer

Okazakifragment

Repairing DNARepairing DNA DNA polymerases proofread newly made

DNA Replacing any incorrect nucleotides

DNA polymerases proofread newly made DNA Replacing any incorrect nucleotides

Repairing DNARepairing DNA DNA polymerases proofread newly made

DNA Replacing any incorrect nucleotides

In mismatch repair of DNA Repair enzymes correct errors in base pairing

DNA polymerases proofread newly made DNA Replacing any incorrect nucleotides

In mismatch repair of DNA Repair enzymes correct errors in base pairing


Nuclease

DNAligase

A thymine dimerdistorts the DNA molecule.1

A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.

2

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

3

DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.

4

DNA Repair

In nucleotide excision repair

Enzymes cut out and replace damaged stretches of DNA

DNApolymerase

Thinking QuestionThinking Question

What is an antioxidiant? Given its name, can you figure out the role

it might play in our bodies? Do you know of any examples of

antioxidants in our diets?

What is an antioxidiant? Given its name, can you figure out the role

it might play in our bodies? Do you know of any examples of

antioxidants in our diets?


End Replication Problem

The ends of eukaryotic chromosomal DNA

Get shorter with each round of replicationEnd of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers andreplacement with DNAwhere a 3 end is available

Primer removed butcannot be replacedwith DNA because

no 3 end availablefor DNA polymerase

Second roundof replication

New leading strand

New lagging strand 5

Further roundsof replication

Shorter and shorterdaughter molecules

5

3

5

3

5

3

5

3

3


End Replication Problem Eukaryotic chromosomal DNA molecules

Have at their ends nucleotide sequences, called telomeres, that postpone the erosion of “real genes” near the ends of DNA molecules

1 µm

End Replication ProblemEnd Replication Problem

If the 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

Some cancer cells have also been found to have functioning telomerase

If the 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

Some cancer cells have also been found to have functioning telomerase

Thinking QuestionThinking QuestionThe topic of cloning is controversial and frequently misunderstood. In a nutshell, the technique of cloning that produced the infamous sheep Dolly used the nucleus of an adult mammary cell from a 6 year old donor sheep and placed it in an empty egg cell. The egg cell was then implanted in a surrogate sheep and allowed to develop as a normal embryo. Knowing what you know about DNA and telomeres, what potential problem do you see with this technique? Anybody know how Dolly died?

The topic of cloning is controversial and frequently misunderstood. In a nutshell, the technique of cloning that produced the infamous sheep Dolly used the nucleus of an adult mammary cell from a 6 year old donor sheep and placed it in an empty egg cell. The egg cell was then implanted in a surrogate sheep and allowed to develop as a normal embryo. Knowing what you know about DNA and telomeres, what potential problem do you see with this technique? Anybody know how Dolly died?

Dolly died and now she's stuffed. Born on July 5th, 1996, Dolly was named for the mammary cell from which she was cloned (in a gentle nod to Dolly Parton), and lived for 6 years at the Roslin Institute in Edinburgh, Scotland in which she was genetically engineered by somatic cell nuclear transfer. She died on Valentine's Day in 2003 and her stuffed remains were put on display 2 months later at the National Museum of Scotland.But if you happen to stand in front of the mute stuffed frame that once was Dolly, there really isn’t much to look at. Dolly is just a sheep, and sheep are not spectacular.

http://www.nms.ac.uk/dollythesheep_1.aspx

http://www.nms.ac.uk/dollythesheep_1.aspx

http://www.ravishingbeasts.com/ungulates/2007/11/28/dolly-the-sheep.html

http://www.ravishingbeasts.com/ungulates/recommend/1055842

Dolly obituaryDolly obituary On 14 February 2003, Dolly was euthanised because of a progressive lung

disease and severe arthritis.[16] A Finn Dorset such as Dolly has a life expectancy of around 11 to 12 years, but Dolly lived to be only six years of age. A post-mortem examination showed she had a form of lung cancer called Jaagsiekte,[17] which is a fairly common disease of sheep and is caused by the retrovirus JSRV.[18] Roslin scientists stated that they did not think there was a connection with Dolly's being a clone, and that other sheep in the same flock had died of the same disease.[16] Such lung diseases are a particular danger for sheep kept indoors, and Dolly had to sleep inside for security reasons.

Some have speculated that a contributing factor to Dolly's death was that she could have been born with a genetic age of six years, the same age as the sheep from which she was cloned.[19] One basis for this idea was the finding that Dolly's telomeres were short, which typically is a result of the ageing process.[20][21] The Roslin Institute have stated that intensive health screening did not reveal any abnormalities in Dolly that could have come from advanced ageing.[19]

On 14 February 2003, Dolly was euthanised because of a progressive lung disease and severe arthritis.[16] A Finn Dorset such as Dolly has a life expectancy of around 11 to 12 years, but Dolly lived to be only six years of age. A post-mortem examination showed she had a form of lung cancer called Jaagsiekte,[17] which is a fairly common disease of sheep and is caused by the retrovirus JSRV.[18] Roslin scientists stated that they did not think there was a connection with Dolly's being a clone, and that other sheep in the same flock had died of the same disease.[16] Such lung diseases are a particular danger for sheep kept indoors, and Dolly had to sleep inside for security reasons.

Some have speculated that a contributing factor to Dolly's death was that she could have been born with a genetic age of six years, the same age as the sheep from which she was cloned.[19] One basis for this idea was the finding that Dolly's telomeres were short, which typically is a result of the ageing process.[20][21] The Roslin Institute have stated that intensive health screening did not reveal any abnormalities in Dolly that could have come from advanced ageing.[19]

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

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

Key Points of Chapter 16Key Points of Chapter 16 DNA is the genetic material Many enzymes work together in DNA

replication and repair

DNA is the genetic material Many enzymes work together in DNA

replication and repair

Chapter 16 DNA Synthesis. Extra Credit – due by Jan. 24, 2014 Up to +24 points added to lowest...

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