Topic A: DNA and Replication

Question 1

What are the 4 key criteria genetic material must possess?

Answer 1

1. Must encode information. As in it must contain the information necessary to make an entire organism
2. Transmission. Must be passed from parent to offspring
3. Replication. Must be copied in order to be passed from parent to offspring, and must be able to self-replicate.
4. Variation. must be able to adapt to changing environments.

Question 2

what are the 4 nitrogenous bases DNA is comprised of? RNA? How do they match?

Answer 2

DNA: Adenine and Thymine, and Cytosine and Guanine

RNA: Adenine and Uracil, Cytosine and Guanine.

Question 3

What are the Purines? Pyrimidines?

Answer 3

Purines: Double ringed, Adenine and Guanine

Pyrimidines: single ringed, cytosine and Thymine (in DNA) or Uracil (in RNA)

Question 4

What are the 3 major components of DNA?

Answer 4

• nitrogenous bases
• pentose/5-carbon sugar
• phosphate group

Question 5

What is the pentose sugar in DNA? RNA?

Answer 5

DNA: Deoxyribose
RNA: Ribose

Question 6

What does it mean when DNA has directionality?

Answer 6

Nucleic acid synthesis always happens from 5’ to 3’. The 5’ end starts with the phosphate group, and the 3’ end starts with the hydroxide (OH) group.

Question 7

What are the main differences between DNA and RNA?

Answer 7

DNA (deoxyribonucleic acid):
- Double stranded sugar phosphate, whose base pairs are cytosine and guanine, and thymine and adenine.
- DNA also has a single hydrogen at the 2’ location on the pentose sugar

RNA (ribonucleic acid):
- Single stranded sugar phosphate whose base pairs are cytosine and guanine, and uracil and adenine.
- RNA has an OH in the 2’ location on the pentose sugar.

Question 8

What did Rosalind Franklin determine about the structure of DNA?

Answer 8

• determined DNA must have a helical structure and that it must be double stranded
• also that the diameter of the helix is 20 angstroms wide and there are 10 bases per 360 degree turn of the helix.

Question 9

What did Erwin Chargaff determine about the structure of DNA? What is Chargaffs rule?

Answer 9

• determined that Adenine and Thymine percentages in a variety of organisms were the same, and guanine and cytosine where the same.
• thus he determines that the amount of Guanine must be the same amount as cytosine, and the amount of thymine must be the same amount as adenine, and thus G+A=C+T, because A=T and G=C.

Question 10

What are the 3 models of DNA replication?

Answer 10

1. Conservative model
   - takes the parental model and forms newly synthasized DNA, one daughter which is formed from the parental and one daughter that is all new.
2. Semiconservative model
   - takes the original model, separates the two strands by breaking the hydrogen bonds, creating daughter DNA that is half old half new.
3. Dispersive model:
   - breaks the phosphodiester bonds within a strand, making daughter DNA that is fragmented half old half new.

Question 11

what model of DNA replication was accepted? Why?

Answer 11

Semiconservative model!
- this is because splitting the bonds between the two strands is easer, as the base pairs are only held together with weak hydrogen bonds, and not the strong phosphodiester bonds that hold adjacent nucleotides together on the same strand.

Question 12

What is DNA composed of? How are the strands orientated?

Answer 12

Two polynucleotide strands/chains that form a double helix, and are anti-parallel to each other.

Question 13

“The deoxyribose sugar and the phosphate groups form the ______ of the helix, while the nitrogenous bases are orientated towards the _____ of the helix”

Answer 13

“The deoxyribose sugar and the phosphate groups form the BACKBONE of the helix, while the nitrogenous bases are orientated towards the INTERIOR of the helix”

Question 14

Nitrogenous bases on opposing pairs hold each other via what kind of bond? How many are between adenine and thymine? Guanine and cytosine?

Answer 14

Hydrogen bonds!
- there are 2 hydrogen bonds between adenine and thymine, and 3 hydrogen bonds between guanine and cytosine.

Question 15

Adjacent nucleotide on the same polynucleotide strand are joined to each other by what kind of bonds?

Answer 15

• phosphodiester bonds

Question 16

How did scientists determine the semi-conservative model was correct? What are the materials they used for the experiment?

Answer 16

• The Messelson-stahl experiment, 1958
• By using E.coli, Heavy nitrogen (N15) and light nitrogen (N14), and a CsCI gradient centrifugation (separates molecules based on density) they were able to establish which mechanism of replication occurred, based on how the DNA settled in the test tube due to density.

Question 17

describe the details to the Messelson-Stahl experiment

Answer 17

• Grow the E. coli bacteria in the N15 (heavy nitrogen) medium. This leaves the E. coli to build up the DNA so it is all heavy.
• Sample at 0 mins
• Transfer the bacteria into N14 (light nitrogen) medium. Bacteria growth continues
• Sample at 20 mins (after one round of replication)
• Sample taken at 40 mins (after two rounds of replication)
• After replication, the DNA would have synthesized new DNA, incorporating the light nitrogen
• Based on the samples at 20 and 40 minutes (1 and 2 rounds of replication) we can see which replication model is correct!

Question 18

How does DNA replication start? where is the site of replication and what ‘factors’ are necessary?

Answer 18

• DNA replication starts are the Ori C site.
• while eukaryotes have multiple Ori C sites, prokaryotes only have 1.
• it is about ~275 bp long
• needs 3 AT-rich sites and 5 DnaA boxes.

Question 19

What is Ori C?

Answer 19

• origin site of DNA replication.

Question 20

What is the goal of replication initiation?

Answer 20

• to separate the 2 polynucleotide strands so they can each be replicated, and be used as a building block for DNA polymerase

Question 21

What are the steps to DNA replication initiation, up to the separation of the strand?

Answer 21

• DnaA proteins bind to DnaA box sequences and to each other. This causes the region to wrap around the DnaA proteins and separates the AT-rich region
• As a result of DnaA protein binding, the torsional stress results in denaturation or melting of the DNA duplex.
• Two DNA helicases (DnaB proteins) bind to the origin. They move in opposite directions from each other, separating the DNA in both directions creating 2 replication forks. (Replication ‘bubble’ is comprised of two replication forks
• Separates the rest of the strand.

Question 22

Why are AT regions seperated during DNA replication? why are the necessary for Ori C sites?

Answer 22

-AT-region is the area that is separated because AT nitrogenous bases only have 2 hydrogen bonds binding them, rather than CG’s 3 hydrogen bonds. Thus, AT is easier to separate.

Question 23

What is DnaA?

Answer 23

• Protein that during DNA replication initiation, binds to the DnaA box sequence, causing the region to wrap around the DnaA proteins and separates the AT rich region.

Question 24

What are DNA helicases?

Answer 24

• also called DnaB proteins
• two working together will bind to the origin site in DNA replication and move in opposite directions from each other, separating the DNA creating 2 replication forks

Question 25

What are single stranded binding proteins?

Answer 25

• (SSBP)
• in DNA replication, stops the base pairs from joining again, keeping them separated from each other.

Question 26

What is Topoisomerase?

Answer 26

• It is an enzyme in DNA replication that relieves the supercoiling caused by the helicase.

Question 27

What is Primase?

Answer 27

• RNA polymerase, synthesizes RNA

Question 28

What is DNA polymerase 3?

Answer 28

• Primary enzyme responsible for the synthesis of new DNA
• Called the “polymerase” function if it is synthesizing new DNA, ALWAYS works from 5’-3’ for synthesizing new DNA
• Called the “exonuclease” function if it is working 3’-5’, essentially proofreads and edits the DNA if mistakes are made

Question 29

DNA polymerase cannot synthesize DNA from scratch. It needs a ________, and help from _____________

Answer 29

DNA polymerase cannot synthesize DNA from scratch. It needs a 3’ OH group to extend from and help from primase.

Question 30

What is nuclease? The two types?

Answer 30

• enzymes that degrade nucleic acids, either DNA or RNA
  Exonuclease enzymes: able to remove nucleotides from terminal ends of polynucleotides strands, but cannot break anywhere along the DNA strand.
  Endonuclease enzymes: able to break DNA anywhere along the strands, not restricted to only the ends.

Question 31

What are Okazaki fragments?

Answer 31

• short sequences of DNA nucleotides which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.
• Ligase forms a new phosphodiester bond making a continuous strand.

Question 32

How are Okazaki fragments linked together?

Answer 32

• DNA polymerase 3 elongates RNA primers with new DNA
• DNA polymerase 1 removes RNA at 5’ end of neighboring fragment and fills gap
• DNA ligase connects adjacent fragments.

Question 33

DNA can only be synthesized in one direction:

Answer 33

from 5’ to 3’

Question 34

What is a lagging strand?

Answer 34

-Strand in DNA replication that is composed of DNA fragments, synthesized from Okazaki fragments.

Question 35

What is the leading strand?

Answer 35

• strand in DNA replication that is synthesized in a continuous fashion