Lecture 10b

Question 1

What are the three initial models of DNA replication?

Answer 1

1. Semi-conservative: the double helix of each daughter
   DNA molecule contains one strand from the original DNA molecule and one newly synthesized strand
2. Conservative: the parent DNA molecule is conserved, and a single daughter double helix is produced consisting of two newly synthesized strands
3. Dispersive: daughter molecules consist of strands each
   containing segments of both parental DNA and newly synthesized DNA

Question 2

Describe the experiment that demonstrated DNA replication is semi-conservative.

Answer 2

Allow parental DNA molecules containing nucleotides of one density to replicate in medium containing nucleotides of different density.
Experiment: Grow E coli in medium containing heavy 15N isotope so that it is incorporated in nitrogenous bases –> Cells moved into medium with light 14N –> cell division –> DNA isolated and separated based on density

Question 3

How was DNA separated by density?

Answer 3

Cesium chloride gradient centrifugation: CsCl is spun in centrifuge at high speed for hours which creates gradient of density in the tube. Larger density at the bottom. DNA is centrifuged with CsCl and forms band at position identical with it’s density.

Question 4

What are the band position predictions for each model?

Answer 4

SC: 1st gen - middle, 2nd gen - middle and top
C: 1st gen - bottom and top, 2nd gen - bottom and top
D: 1st gen - middle, 2nd gen - between middle and top

Question 5

What are the 3 activities of DNA pol I?

Answer 5

1. Polymerase activity in 5’ to 3’ direction
2. 3’ to 5’ exonuclease activity to remove mismatched bases
3. 5’ to 3’ exonuclease activity to degrade single strands of DNA or RNA (replaces RNA primer)
   DNA pol I is much slower than pol III

Question 6

Describe DNA replication in prokaryotes.

Answer 6

• One ori per chromosome
• Occurs in cytoplasm
• DnaA is a protein that recognizes the oriC and pulls apart strands (opens helix)
• Helicase then unzips the rest of the DNA. Helicase forms complex with replisome
• Two replication forks
• Bidirectional
• Forms theta structure

Question 7

What is a replisome?

Answer 7

“Molecular machine” that carries out DNA replication. It is composed of DNA pol I, DNA pol III, helicase, gyrase, ligase, SSB, and other proteins.

Question 8

What are helicases?

Answer 8

Enzymes that disrupt the hydrogen bonds that hold the two strands of the double helix together. It unzips the DNA helix ahead of DNA synthesis.

Question 9

What are single-strand binding proteins (SSB)?

Answer 9

Stabilize unwound DNA by binding to single-stranded DNA and preventing the duplex from re-forming.

Question 10

What are topoisomerases?

Answer 10

Relax supercoiled DNA by breaking either a single DNA strand or both strands, which allows DNA to rotate into a relaxed molecule. Topoisomerases finish by rejoining the strands of the now relaxed DNA molecule.

AKA DNA gyrase

Question 11

Describe DNA replication in eukaryotes.

Answer 11

• Multiple ori per chromosome
• Multiple replication bubbles (thus multiple replication forks)
• YEAST: Presence of origin recognition complex (ORC) recruits Cdc6 and Cdc1 proteins. All together, they recruit helicase and replisome assembles. DNA replication is restricted to the S phase in cell cycle because G1 is the only time Cdc6 and Cdc1 are produced. They are destroyed after synthesis.

Question 12

What what direction is DNA made?

Answer 12

5’ to 3’

Question 13

What end are nucleotides added to?

Answer 13

3’ OH

Question 14

What is the difference between the leading and lagging strand?

Answer 14

Leading: made continuously, 5’ to 3’ synthesis occurs in same direction as fork migration

Lagging: made discontinuously, 5’ to 3’ synthesis occurs in opposite direction as fork migration, creates series of small okazaki fragments (fragments are longer in prok than euk)

Question 15

What are the 6 most important enzymes of DNA replication?

Answer 15

Helicase, SSB, primase, DNA pol III, DNA pol I, DNA ligase

Question 16

How is the RNA primer made?

Answer 16

Primase (type of RNA pol) makes a short RNA complementary to DNA which is later removed by pol I. Primer is needed because DNA pol can extend DNA chain but cannot start it. Each okazaki fragment requires a primer.

Question 17

What is the function of DNA pol III?

Answer 17

Adds bases to existing 3’ OH. It makes DNA 5’ to 3’ and moves on the template DNA strand 3’ to 5’. Has exonuclease activity (can backspace on mismatches)

Question 18

What are the components of the DNA pol III holoenzyme?

Answer 18

2 catalytic cores + beta clamps + accessory proteins. One core handles synthesis of leading strand and the other handles synthesis of lagging strand.

Question 19

What is the function of DNA ligase?

Answer 19

Joins the 3′ end of the gap-filling DNA to the 5′ end of the downstream Okazaki fragment

Question 20

What what point is DNA replication terminated?

Answer 20

When replication forks moving in opposite directions meet

Question 21

What is DNA pol I not capable of?

Answer 21

It cannot replace the primer at the very end of linear DNA molecules because there is no upstream.

Question 22

What are telomeres? Why are they necessary?

Answer 22

Telomeres are specialized structures at the ends of chromosomes that contain tandem repeats of a short DNA sequence that is added to the 3′ end by the enzyme telomerase. Telomeres stabilize chromosomes by preventing the loss of genomic information after each round of DNA replication and by associating with proteins to form a cap that “hides” the chromosome ends from the cell’s DNA-repair machinery.

Question 23

What is the function of telomerase?

Answer 23

Adds repeats onto telomere if needed. It uses a small RNA as a template for the repeat. Telomerase is a polymerase and matches DNA bases to its RNA (technically a RT). The overhang of repeats it creates can be tucked into a protective cap to prevent the end from being mistaken for double stranded break.

Question 24

Where is telomerase active?

Answer 24

Germline cells not somatic cells. Therefore, telomeres are gradually lost in somatic cells with every division. If telomerase is active in somatic cells it will cause cancer.