15 DNA and the Gene: Synthesis and Repair

Question 0

Describe the Hershey-Chase experiment

Answer 0

1) Label viruses: grow some viruses in the presence of P-32 for radioactive DNA and some with S-35 for radioactive protein.
2) Infect bacteria: Allow viruses with labelled DNA to infect a culture of E. coli cells.
3) Agitate Cultures: in kitchen blenders to separate virus capsules from bacterial cells.
4) Centrifuge Solution: to force bacterial cells into a pallet.
5) Record: location of radioactive labels.

Question 1

What is DNA?

Answer 1

Deoxyribonucleic acid: a double helix molecule compose of four varying nucleotides with a deoxyribose sugar; that encodes for genetic information

Question 2

What were the results and conclusion of Hershey-Chase experiment?

Answer 2

Results:

1) radioactive DNA is in pallet.
2) radioactive protein is in solution.

Conclusion: viral genes consist of DNA

Question 3

Who proposed the double helix structure of DNA?

Answer 3

Watson and Crick

Question 4

What does each deoxyribonucleotide consist of?

Answer 4

1) phosphate group
2) deoxyribose sugar
3) nitrogenous base

Question 5

How do nucleotides link together?

Answer 5

Link together into a polymer by forming diester bond between the hydroxyl group on the 3’ carbon and the phosphate group on the 5’ carbon

Question 6

What are the four nitrogenous bases?

Answer 6

Adenine
Thymine
Guanine
Cytosine

Question 7

What are the base pairs?

Answer 7

Adenosine - Thymine

Guanine - Cytosine

Question 8

What are the three alternative hypothesis about DNA synthesis?

Answer 8

1) Semiconservative replication
2) Conservative Replication
3) Dispersive Replication

Question 9

What is the Semiconservative replication theory?

Answer 9

If the old, parental strands of DNA separated, each could then be used as a template for the synthesis of a new, daughter strand.

Each new daughter DNA molecule would consist of one old strand and one new strand.

Question 10

What does the Conservative replication theory state?

Answer 10

If the bases temporarily turned outward so that complementary strands no longer faced each other, they could serve as a template for the synthesis of an entirely new double helix.

Would result in an intact parental molecule and a new daughter molecule consisting of entirely newly synthesized strands

Question 11

What does the dispersive replication theory state?

Answer 11

If the parental double helix were cut wherever one strand crossed over another and DNA was synthesized in short sections by extending each of the cut parental strands to the next strand crossover, then there would be a mix of new and old segments along each replicated

Stretches of old DNA would be interspersed with new DNA down the length of each daughter cell.

Question 12

What is the Meselson-Stahl Experiment?

Answer 12

An experiment that was used to determine how DNA is synthesized. Choosing between Semiconservative, conservative and dispersive replication.

Question 13

Outline the process of the Meselson-Stahl Experiment

Answer 13

1) grow E. colin cells in medium with N-15 as a sole source of nitrogen for many generations. Collect sample and purify.
2) transfer cells to medium containing N-14. After cells divide once, collect sample and purify DNA.
3) after cells have divided a second time in N-14 medium, collect sample and purify.
4) centrifuge the three samples separately. Compare the locations of the DNA bands in each sample.

Question 14

Outline the predictions of the Meselson-Stahl experiment

Answer 14

Predictions, after 2 generations:

• Semiconservative: 1/2 low density, 1/2 intermediate-density DNA
• Conservative: 1/4 high density DNA, 3/4 low density DNA
• Dispersive: all intermediate density DNA (hybrid)

Question 15

Outline the results and conclusion of the Meselson-Stahl experiment

Answer 15

Results: after two generations 1/2 were low density (N-14) and 1/2 were intermediate density DNA (hybrid)

Conclusion: data from generation 1 conflict with conservative replication hypothesis. Data from generation 2 conflict with dispersive replication hypothesis. Therefore, replication is Semiconservative

Question 16

In what direction can DNA polymerase add deoxyribonucleotides to DNA?

Answer 16

Can only add to the 3’ carbon, therefore, 5’ –> 3’ direction

Question 17

Is DNA synthesis endergonic or exergonic?

Answer 17

Most polymerization reactions are endergonic (require energy input), however, DNA synthesis is exergonic (it releases energy).

The monomers that are used in DNA synthesis are deoxyribonucleoside triphosphates (dNTP). The release of the phosphate groups release enough energy for the reaction to occur.

Question 18

DNA is ————- — that is, it occurs in both directions at the same time

Answer 18

Bidirectional. Therefore, replication bubbles grow in two directions as DNA replication proceeds.

Question 19

Bacterial chromosomes have a —— —— of replication

Answer 19

Single origin

Question 20

Eukaryotic chromosomes have ——– ——- of replication

Answer 20

Multiple origins

Question 21

How is the helix opened and stabilized?

Answer 21

1) the enzyme DNA helicase breaks the hydrogen bonds between base pairs causing the two strands to separate
2) single-strand DNA-binding proteins (SSBPs) attach to the separated strands and prevent them from snapping back into a double helix.
3) unzipping process creates tension because of twisting forces
4) the enzyme, topoisomerase, cuts DNA, allowing it to unwind, and rejoins it ahead of the advancing replication fork. - releases tension.

Question 22

What is a primer?

Answer 22

• A strand of a few nucleotides long that is bonded to the template strand
• provides DNA polymerase with a free 3’ hydroxyl group that can combine with an incoming deoxyribonucleotide to form phosphodiester bond.

Question 23

How is the leading strand synthesis?

Answer 23

1) an enzyme primase synthesizes a short stretch of RNA that acts as a primer to DNA polymerase.
2) DNA polymerase begins working in a 5’ —> 3’ direction and adds deoxyribonucleotides to the complementary strand
3) as the polymerase moves along the DNA molecule, a doughnut-shaped molecule, called the sliding clamp, holds the enzyme in place on the template strand.
4) the enzymes product is called the leading strand, or continuous strand, because it leads into the replication fork and is synthesized continuously.

Question 47

How is the lagging strand synthesize?

Answer 47

1) Lagging strand is anti-parallel to the leading strand.
2) Since DNA synthesis occurs in a 5’—>3’, the replication of the lagging strand occurs away from the replication fork and so must be done in fragments (Okazaki fragments).
3) Primase synthesizes RNA primer.
4) first fragment synthesized by DNA polymerase
5) process repeats for multiple fragments
6) Primer replaced with deoxyribonucleotides by DNA polymerase 1 in 5’—>3’
7) Gap closed: in sugar-phosphate backbone by DNA ligase

Question 48

What is the region at the end of a eukaryotic chromosome?

Answer 48

Telomere

Question 49

What is the end replication problem?

Answer 49

• on the lagging strand, primase adds an RNA primer close to the tip of the chromosome
• DNA polymerase synthesizes the final Okazaki fragment removing the primer
• DNA polymerase is unable to add more DNA on the tip of the chromosome without the primer. Leaving a short single stranded end.
• the single-stranded DNA at the end of the chromosome is eventually degraded, which results in the shortening of the chromosome.
• if process continues, after each replication, chromosomes become 50-100 nucleotides shorter

Question 51

What enzyme repairs the telomeres?

Answer 51

Telomerase

Question 52

How does telomerase repair the telomeres?

Answer 52

1) telomeres do not contain genes but are made of short stretches of bases that are repeated over and over. In Humans TTAGGG is repeated.
2) the unreplicated segment forms a single-stranded overhang
3) telomerase binds to the overhang and begins DNA synthesis. Done by a portion of telomerase that is RNA
4) telomerase synthesizes DNA in a 5’—>3’ and catalyses repeated additions of the same sequence to the end of the strand
5) once the single-stranded overhang on the parent strand is lengthened the normal enzymes of DNA synthesize a complementary strand
6) lagging strand becomes slightly longer

Question 53

How does DNA polymerase correct mistakes in DNA synthesis?

Answer 53

1) DNA polymerase III proofreads, the positioning of the incorrect nucleotide provides poor substrate for polymerase to extend due to different geometry.
2) polymerase will only add the next nucleotide when the mistake is fixed. (usually)
3) the ε subunit in polymerase acts as an exonuclease and removes the wrong nucleotide by 3’—>5’ direction.
4) the polymerase then adds the correct nucleotide.

Question 54

What is mismatch repair?

Answer 54

Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete.

• proteins recognize the mismatched pair, remove a section containing the incorrect base and fill in the correct base.
• chemical marks on the older strand allow enzymes to distinguish the original from the newly synthesized.

Question 55

What can damage DNA?

Answer 55

-radiation (UV, X-rays, gamma ray)
-chemicals
—> hydroxyl radicals
—> aflatoxin B1 found in moldy peanuts and corn
—> benzo[α]pyrene in cigarette smoke

Question 56

How does UV light damage DNA?

Answer 56

• UV in sunlight or tanning booths can break the bonds between basepairs with thymine.
• the same energy may lead to adjacent thymines to bind together producing a thymine dimer (kink)
• kink stalls DNA replication (even prevent it)

Question 57

What is nucleotide excision repair?

Answer 57

Nucleotide excision repair fixes thymine dimers and many other types of damage to DNA.

1) damage region is recognized
2) enzyme removes a segment defective sequence
3) intact DNA strand provides a template for synthesis of corrected strand
4) 3’ hydroxyl group acts as primer
5) DNA ligase links the newly synthesized DNA to undamaged DNA.

Question 58

__________ __________ is a rare autosomal recessive disease in humans. Individuals with this condition are extremely sensitive to UV light. What are the symptoms?

Answer 58

Xeroderma Pigmentosum (XP)

Symptoms:
skin lesions - rough, scaly patches, irregular dark spots after slight exposure.

Due to limited ability of nucleotide excision repair