DNA replication

Question 1

Define helicase

Answer 1

The enzyme that unzips the DNA for replication

Question 2

Define SSBP protein (AJT photo)

Answer 2

A protein that binds to the single-stranded DNA to avoid hydrogen bonds between the two diff strands
- prevents closing

Question 3

(REVIEW) why is a DNA strand 5’ to 3’ and 3’ to 5’ (aka why are they anti-parallel?) [add another photo]

Answer 3

For the 5’ to 3’:
- The 5th carbon (phosphate atom) bonds to the third carbon of another

For the 3’ to 5’:
- no “horizontal flip” (my words) — MUST rotate
- to make the nitrogenous bases face each other
- make the hydrogen bonds
- The third carbon (the phosphate atom bonds to the 5th carbon

Question 4

Describe the process of DNA replication

Answer 4

• Helicase unwinds
• SSBP attaches to nucleotides to avoid closing
• primer attached to nucleotides to let the DNA Polymerase know where to start
• DNA polymerase continuous adding with leading strand (old: 3’ to 5’, new: 5’ to 3’)
• Lagging opposite direction (old: 5’ to 3’. new: 3’ to 5’)
• DNA polymerase moves in intervals bc of it
• okazaki fragments added to DNA replication
• DNA ligase (poly I) “glue” the okazaki frags together by adding a nucleotide in between
• then two semi-conservative daughter DNA is formed

Question 5

Describe DNA polymerase III [2]

Answer 5

An enzyme that adds nucleotides to the two old DNA strands during replication
- moves in 5’ to 3’ direction
- therefore, leading strand OK with direction
- however, NOT ok with lagging strand

Question 6

Define RNA primer

Answer 6

a short genetic sequence of RNA that tells the DNA polymerase where to start

Question 7

Define primase (?)

Answer 7

the enzymes that synthesize RNA primers (from free nucleotides?)

Question 8

What is the direction of the semi-conservative model (aka old strand and new strand)

Answer 8

For leading:
- old strand: 3’ to 5’
- new strand: 5’ to 3’

For lagging:
- old: 5’ to 3’
- new: 3’ to 5’

Question 9

Define DNA replication

Answer 9

DNA sequence is copied during S-Phase of cell cycle

Question 10

Define DNA gyrase

Answer 10

• enzyme that stabilizes DNA structure as it unwinds
• why?: the compression of the wound/ not yet unwinded parts of the DNA unwinding

Question 11

Define DNA polymerase III

Answer 11

• enzyme
• adds nucleotides in the 5’ to 3’ direction
• (both lagging and leading)

Question 12

define DNA ligase

Answer 12

joins okazaki fragments on lagging strand
- seals the nicks

Question 13

Describe the PCR

Answer 13

• synthetic (artificial) method of lengthening specific seuqences of DNA
• useful when theres small amount of DNA available (e.g. crime scenes)
• Taq DNA polymerase enzyme is used for PR

Question 14

Describe Taq DNA polymerase

Answer 14

• used for PCR
• from thermus acquaticus, heat resistant bacterium
• therefore, resists denaturation at high temperatures
• copies up 1000 nucleotides at a minute

Question 15

Describe the PCR process

Answer 15

1.) Denaturation
- DNA sample heated to 95*C
- to break hydrogen bonds and separate to 2 strands

2.) Annealing
- DNA sample cooled
- primers attach to the opposite ends of target seq

3.) Elongation (70C - 75C)
- heat-tolerant/ Taq DNA polymerase copies strands

Question 16

What determines if a DNA strand is 5’ to 3’ or 3’ to 5’

Answer 16

which carbon in the pentose sugar is exposed

Question 17

What is the role of the RNA primer?

Answer 17

• To let the DNA polymerase know where to start
• By adding a short nucleotide sequence wherein other nucleotides can bond to (?)

Question 18

In which direction and where can the DNA helicase unwind?

Answer 18

Anywhere and both directions as long as its an A-T rich region

Question 19

Why is the 3’ carbon the ONLY available carbon for bonding in DNA?

Answer 19

Because it has one bond left
- available for phopshodiester bond

Question 20

Give the alternative name for PCR

Answer 20

Thermocycle

Question 21

Define “vitro”

Answer 21

outside
- e.g.: PCR = vitro DNA replication

Question 22

What is the temperature in the denaturation process of PCR?

Answer 22

90-95*C

Question 23

Give the temperature for annealing phase of PCR

Answer 23

50-60*C

Question 24

Give the temperature for the elongation phase of PCR

Answer 24

• 70-75*C
• (for the Taq Polymerase)

Question 25

Give the purpose of the “Sanger Sequence”

Answer 25

- To know the full genetic sequence

Question 26

What makes Sanger Sequence different from PCR?

Answer 26

the use of ddNTP

Question 27

Define ddNTP

Answer 27

dideoxyribonucleic tri-phosphate - deoxyribose sugar without an Oxygen atom at 3’ - attached to 3 phos

Question 28

Give the different types of ddNTP

Answer 28

1.) ddATP 2.) ddTTP 3.) ddCTP 4.) ddGTP

Question 29

What does ddNTP do?

Answer 29

it stops the (further) genetic sequencing

Question 30

How does ddNTP terminate genetic sequencing?

Answer 30

It removes the oxygen carbon of 3’ - removes the bonding ability due to removal of oxygen’s additional bond

Question 31

What will happen to the DNA sample because of ddNTP?

Answer 31

Instead of 1 long DNA strand, we get multiple of diff lengths

Question 32

Describe the process of Sanger Sequencing

Answer 32

1.) DNA sample - DNA strand split into 4 test tubes - 1 ddNTP each - attaches and stops the sequencing at random pts 2.) Gel electrophoresis - separation of DNA by size - in wells - electric current: law of attraction, poles, mvmt. - most neg: most long -> most positive: least long 3.) Analysis - read from the bottom up - determine the sequence

Question 33

How do the *single strand binding proteins* prevent repannealing

Answer 33

The pull away the backbones of the strands which breaks the hydrogen bonds

Question 34

What does the exonuclease do?

Answer 34

The exonuclease removes the RNA primers because of their different structure AND replaces them with DNA

Question 35

What is the function of DNA ligase

Answer 35

To seal the nicks

Question 36

Define dNTP

Answer 36

deoxyribonucleos*ide*

Question 37

Distinguish nucleotide and dNTP

Answer 37

**dNTPs:** - from the *cytoplasm* - used by *DNA polymerase III* to make new strands - *Tri-phosphate* - *loses two phosphate* because of hydrogen and phosphodiester *bond* - becomes nucleotide **Nucleotide:** - One phosphate only

Question 38

Why can’t lytic cycle become lysogenic?

Answer 38

Because of lysis (cell burst)

Question 39

Discuss the evidence provided by viral structure and function that suggests they evolved from living organisms

Answer 39

- complexity in structure of some viruses would suggest bacteria ancestor - e.g. maybe from parasitic bacteria - retroviruses and some eukaryotic cells w/ reverse transcriptase - genetic material moved out of cells - obligate parasites, couldn’t exist without cells - viruses have similar genetic material as cells (DNA and RNA)

Question 40

Outline the stages in the production of mRNA by transcription

Answer 40

- DNA is unwound by **RNA POLYMERASE**,, breaks the hydrogen bonds between the DNA nucleotides - new nucleotides **complementary bound** to the **anti-sense strand** by RNA polymerase, *transcribing the DNA in a 5' to 3' direction.* -* A to U, C to G* - **newly synthesized mRNA detaches from anti-sense/template strand** strand and unwinds

Question 41

Give the alternative name for exonuclease

Answer 41

DNA polymerase I

Question 42

How do dNTPS become Nucleotides?

Answer 42

They bond to others nucleotides and lose 2 phosphate atoms for the hydrogen bond and the phosphodiester bond)

Question 43

Give the temperatures for PCR (BOOK)

Answer 43

1.) Heating: 90-95*C 2.) Cooling: 54*C 3.) Elongation: 72*C optimum for TAQ DNA polymerase

Question 44

Outline the process of DNA profiling [4]

Answer 44

- DNA is obtained from material/individual (e.g. crime scene, hair, blood, mouth, swab, etc.) - *tandem repeats are copied by PCR ~13 times min. - gel electrophoresis separates by lengths - DNA patterns always the same and is unique to individual - [insert application here]

Question 45

Explain Chargaff Experiment (brief)

Answer 45

- Main Findings in Support of: **Complementary base pairing** + ** DNA IS THE GENETIC MATERIAL** - BEFORE: tetranucleotide — equal nitro base concentration. - DURING EXP: Sampled the nucleotide concentration of different orgs + viruses - AFTER: A-T and C-G very similar concentrations, DNA has variation