Theme 1C

Question 1

What is conservative DNA replication?

Answer 1

Template strands are preserved throughout replication, after replication both daughter strands pair up. There is a conserved original strand

Question 2

What is dispersion DNA replication?

Answer 2

Daughter strands have a mixture of parental and newly synthesized DNA (a random nucleotide collection)

Question 3

What is semiconservative DNA replication?

Answer 3

Each daughter strand remains paired with its complementary parental strand

Question 4

What was the basis of the Meselson and Stahl experiment?

Answer 4

Track parental and newly-synthesized DNA strands over several generations using nitrogen isotopes

Question 5

What were the steps of Meselson and Stahl’s experiment?

Answer 5

1.) N14 is lighter and less dense isotope than N15. They put E.coli with N15 which would mean nucleotides would contain 100% N15 in their nitrogenous bases

2.) DNA was isolated and centrifuged creating a gradient (lighter things on top, heavier on the bottom). DNA settles in a position based on density

3. Transferred E.coli to N14, sampled DNA after two rounds of replication which proved semiconservative replication

Question 6

What is DNA polymerase?

Answer 6

• An enzyme
• Adds nucleotides to the new strand at the 3’ OH end of a double stranded molecule
• Cannot synthesize from template strand, requires an RNA primer with a 3’ OH for synthesis
• Single active site that catalyzes four reactions (dATP, dCTP, dGTP, dTTP)

Question 7

Where does DNA replication start in Eukaryotes?

Answer 7

Multiple points along the length of each DNA molecule. Each starting site is referred to as the origin of replication

Question 8

What happens at the origin of replication?

Answer 8

1. As the two parental strands separate, a replication bubble forms
2. Each replication bubble has two replication forks
3. The replication forks are the site of DNA polymerization and points in the opposite direction and move away from each other as DNA synthesis continues
4. Two replication bubbles moving in opposite direction eventually merge with one another

Question 9

What is helicase?

Answer 9

An enzyme that untwists the double helix at each replication fork. Separates the two “old” template strands. Once helicase has moved past, the separated strands will re-anneal

Question 10

What is single stranded binding protein (SSBP)?

Answer 10

These proteins bind single stranded DNA and prevents them from re-annealing before replication starts. Basically keeps strands separated once helicase has passed.

Question 11

What is Topoisomerase?

Answer 11

An enzyme that releases tension (supercoiling) caused by helicase ahead of the replication fork

Question 12

What is primase?

Answer 12

Enzyme that synthesizes a short RNA primer at oor. Unlike DNA polymerase, it can add RNA nucleotides without existing 3’-OH to a single stranded template.
- Adds 10-20 RNA nucleotides base paired with the template strand forming a primer (DNA/RNA hybrid)

Question 13

What is DNA polymerase 3?

Answer 13

Enzyme that adds dNTPs to 3-OH’. Starts at 3’-OH and ends at RNA primer, so it reads the template strand in the 3’-5’ direction. Yet adds dNTP’s in the 5’-3’ direction

Question 14

What is a DNA sliding clamp?

Answer 14

Stabilizes DNA polymerase so it does not fall off the template strand

Question 15

What is the leading strand?

Answer 15

Elongates continuously and is read in the 3’-5’ direction.

Question 16

What is the lagging strand?

Answer 16

Antiparallel to the leading strand, faces away from the replication fork and towards the oor. Elongates discontinuously in a series of short segments called okazaki fragments

Question 17

What happens when the replication bubbles collide?

Answer 17

One parental template strand and one daughter strand that is a mix of DNA + RNA primers come together

Question 18

What is DNA polymerase 1?

Answer 18

Removes the RNA nucleotides and replaces them with DNA nucleotides. The last dNTP added by 1 will be next to the first dNTP added by 3. These adjacent nucleotides cannot join together however

Question 19

What is DNA ligase?

Answer 19

Seals the “nicks” between the two dNTPs by forming a phosphodiester bond between them

Question 20

What is the replisome?

Answer 20

Everything is clumped together to increase the efficiency of replication

Question 21

What are telomeres?

Answer 21

Non-coding DNA at both ends of linear chromosomes that protect coding DNA. Usually repeats of 5-8 G’s and T’s. Approximately 10k base pairs long, though some will be worn away after each round of replication.

Question 22

What happens when the telomere region is gone?

Answer 22

Cells stop dividing.

Question 23

What is telomerase?

Answer 23

Keeps stem cells immortal, has RNA nucleotides that extends worn out telomeres. Restores shortened telomeres. Usually not present in MOST eukaryotic cells.

Question 24

What are the three main DNA repair mechanisms?

Answer 24

1. Proofreading: DNA polymerase 3 detects mistakes and uses its 3’-5’ exonuclease activity
2. Mismatch repair (MMR): replication errors not covered by proofreading, done by MutS and MutL
3. Base excision repair: DNA glycosylases removes modified base forming AP site, AP endonuclease removes AP site and polymerase repairs damage

Question 25

What are the steps of mismatch repair (MMR)?

Answer 25

- DNA binding proteins MutS and MutL recognize mismatch damage - MutH endonuclease nicks daughter strand several nucleotides away from the mismatch - Exo1 5'-3' excises region of daughter strand surrounding mismatch (snips it off) - DNA polymerase 3 fills the gap and repairs mismatch - The nick left after the gap is sealed by ligase

Question 26

What are the steps for Sanger sequencing?

Answer 26

1. DNA purification (get rid of other cell macromolecules) 2. DNA fragmentation (use restriction enzymes to chop the DNA into smaller pieces) 3. Amplication (use PCR to make hundreds of copies of each fragment) 4. Sequencing (determine the base sequence of each fragment) 5. Assembly (put the fragments back together)

Question 27

What type of nucleotides are used for the technique to sequence DNA?

Answer 27

Nucleotides that lack a 3' hydroxyl group on both the 2 carbon and 3 carbon of pentose sugar (dideoxynucleotides). When ddNTP base pairs with template, DNA polymerase adds it to the growing daughter strand, but then replication stops because additional dNTP's can't be added to the ddNTP

Question 28

What will be the last nucleotide of the new strand?

Answer 28

The ddNTP

Question 29

What is the ORIGINAL Sanger sequencing?

Answer 29

The template DNA is placed into 4 test tubes: with DNA polymerase, DNA primer (base pairs with template fragment and labelled 32P), the four normal dNTP's (one in each tube individually), small amount of one ddNTP (each tube will have different). - Tube is heated so DNA denatures - When tubes cool, primers attach to template DNA and DNA polymerase starts adding nucleotides before re-annealing can happen

Question 30

What is gel electrophoresis?

Answer 30

- A sequencing gel that is a porous matrix - Loaded at one end of the gel - Electric field generated: negative at the top and positive at the bottom - DNA migrates towards positive charge - Smaller particles move faster

Question 31

What happens following original Sanger sequencing?

Answer 31

- Gel is exposed to X rays to visualize DNA - Each lane of the gel represents a different nucleotide - The bands in each lane represent DNA fragments at different lengths (shortest at the bottom, longest at the top)

Question 32

What is modern Sanger sequencing?

Answer 32

- Uses ddNTP's that have fluorescent tags (fluoresce at different wavelengths) - Sequencing can be done in a single test tube (template DNA, primers, all 4 dNTPs, all 4 ddNTPs, polymerase - Following replication, samples sent through an electrophoretic gel column - Shortest sequences reach the bottom of the column first and pass a laser that detects the fluoresce ddNTP - DNA sequence can be determined from the fluoresce pattern

Question 33

What are the requirements for polymerase chain reaction (PCR)?

Answer 33

- Template DNA - DNA primers (one for each strand) - dNTPs - Heat tolerant DNA polymerase (Taq polymerase) - PCR machine (thermocycler)

Question 34

What are the steps in PCR?

Answer 34

1. Heat the sample to 95 degrees 2. DNA strands separate 3. Cool the sample to 55 degrees 4. DNA primers anneal 5. Heat the sample to 72 degrees 6. Taq polymerase synthesizes daughter strands 7. Repeat

Question 35

What is the first way PCR can be applied?

Answer 35

DNA fingerprinting. - We have multiple non-coding sequences called short tandem repeats (STR's) that are very variable - PCR can amplify those regions in a DNA sample

Question 36

What is the second way PCR can be applied?

Answer 36

Phylogeny. - All living organisms have the gene for 16s rRNA (the small ribosomal subunit) - DNA from organism can be isolated and the rRNA gene can be amplified by PCR and then sequences - Compare new sequence to known sequences of other organisms