Lecture 5: DNA structure and replication

Question 1

What were the two strains in the Griffith Experiment and what were their differences?

Answer 1

S strain ( virulent/ killed mice)
R strain( non-virulent mouse lived)

Question 2

What was the process of the Griffiths experiment and what were the results?

Answer 2

S strain injection–> killed mice
R strain injection–> did not kill mice
Heat killed s strain–> did not kill mice
Heat Killed IIS and IIR–> killed mice and created IIIS strain —> the hereditary material of the IIS strain transformed the IIR strain to be virulent

Discovered process of transformation

Question 3

How do mutations change the strains and why cant mutations explain the transformation of the strain?

Answer 3

Mutations can change a strain from non-virulent to virulent(R to S) but not strain number

Mutation can not explain the transformation of the strain as the strain number changed as well from II–> III

Question 4

What was the Avery, McCarty and McCleod Experiment process and what were the conclusions?

Answer 4

Lysed S cells and separated contents into DNA, RNA, Protein, Lipids, and Carbs

Each macromolecule was destroyed separately into separate transformations

The DNA-destroyed extract was the only one to not transform

Results: DNA is the transforming principle

Question 5

What was the process of Hershey-Chase experiment and what were the conclusions?

Answer 5

DNA was radioactively labeled with Phosphorous
Protein was radioactively labeled with sulfur
Both were grown in separate media and injected into bio macrophages
Only Phosphorous was detected in the host cell–> DNA is the hereditary material!

Question 6

Who discovered the structure of DNA? What did they discover about it? What tools did they use to make these discoveries?

Answer 6

Using X-ray diffraction Rosalind Franklin discovered that DNA has a double helix structure. Her images were used by Watson and Crick

Question 7

What type of bonds hold nucleotides together? Strands together?

Answer 7

Phosphodiester bonds between 3’ OH and 5’ Phosphate hold nucletides together

Hydrogen bonds betwen nucleotides of each strand holds them together

Question 8

Why is parallel DNA double helix unstable but antiparalle is not?

Answer 8

Parallel does not allow for complementary base pairing and disrupts partial charges of bases –> preculudes H bonding

Question 9

What is the DNA structure?

Answer 9

Right-handed double helix
complementary strands
Anti-parallel (allow for base pairing)
Base Stacking ( held together by H bonds)–> imparts a twist in double helix creating major and minor groove ( binding proteins bind to both grooves)

3’ OH, 5’ Phosphate

Question 10

What are the three forms of DNA, and what are their differences?

Answer 10

Most organisms have the B-form
Desiccating contions cause B form to change to A form in some bacteria

Bacteriophages have the A-form which is more compact and less prone to damage

Z-form is a left-handed helix ( backbone is zigzag)

Question 11

Why must mutation rates be low but not zero?

Answer 11

Mutations must be low to allow for genetic continuity and most mutations cause harm

Mutations cannot be zero as this would violate darwins first premise (The population will change throughout time as adaptive traits begin to accumulate)

Question 12

What is DNA proofreading? what enzyme is involved? Does it increase or decrease mutations?

Answer 12

DNA proofreading involves DNA polymerase (high fidelity and high specificity):
3’ to 5’ exonuclease activity where it proofreads DNA and removes mutated bases.

Decreases mutations

Question 13

What was the process of the Meselson Stahl Experiment? What did it conclude?

Answer 13

Process:
growth of E.coli in heavy nitrogen (N15)
DNA shows up with density of N15

Then intervals of incorporating heavy and light nitrogen ( Hybrid/intermediate DNA strand with A N15 strand and N14 strand)

Conclusion: DNA is semi-conservative

Question 14

What is the origin of replication, replication bubble, and replication forks?

Answer 14

Origin of replication: where replication begins in prokaryotes and eukaryotes

Replication bubble: bubble between strands result of replication

Replication forks: at each end of replication bubble

Question 14

Suppose the Meselson Stahl experiment showed two bands after one round of replication, one
14/14 band and one 15/15 band. How would you interpret this result?

Answer 14

This would indicated that DNA is fully conservative instead of semi-conservative

Question 15

What was the process and result of the Huberman-Riggs Pulse-chase experiment?

Answer 15

“pulse” radioactive compound to be incorporated into DNA

“chase” it with the nonradioactive version of the compound

results show radioactivity present out of the origin of replication going in both directions

Conclusion: DNA replication is bidirectional

Question 16

Explain replication initiation in bacteria. Use DnaA, DnaB, DnaC, 9-mer, 13-mer, and SSB in your
answer.

Answer 16

1. DNA binds to the 9-mer region of DNA
2. This results in the formation of an open complex at the 13 mers region
3. DNA B delivered to open complex vis DNA C and elicits helicase activity to further separate strands in both directions

Question 17

Explain the order of the three main steps of eukaryotic replication initiation. Use preRC, ORC,
Cdc6, and Ctd1 in your answer.

Answer 17

1. preRC: pre recognition complex of 14 proteins assembles at origin of replication
2. 6 of these proteins act as intiators identifying origin site–> for Origin of Replication Complex(ORC)
3. Cdc6 and Ctd1 bind ORC recruit 8 other proteins to separate the DNA into two strands

Question 18

What is the role of primase in DNA replication? What property of DNA polymerases makes
primase necessary?

Answer 18

Primase adds the RNA primer that allows DNA elongation to begin.

DNA Polymerase III has a sliding clamp mechanism and must attach to a primer due to its low processivity( ability to begin movement on its own) making primase necessary.

Question 19

What is the difference between the leading and the lagging strand

Answer 19

The leading strand is synthesized continuously
The lagging strand is synthesized in Okazaki fragments since dNTPs cannot be added to the 5’ end

Question 20

What is the role of DNA polymerase I in DNA replication? DNA ligase?

Answer 20

DNA Polymerase I removes RNA primer (5’ to 3’ exonuclease activity) and fills gap (5’ to 3’ polymerase activity)

DNA ligase connects Okazaki fragments

Question 21

What is the role of topoisomerases? Why are they needed?

Answer 21

Creating temporary nicks that are later sealed to release the supercoiling caused by helicase activity

Question 22

Explain the end replication problem for linear chromosomes?

Answer 22

When the RNA primer is removed on the lagging strand there is a gap as no elongation can occur on the 5’ end –> resulting in 3’ overhang of parental strand and shortening of chromosomes after each cycle of cell division

Question 23

How does telomerase alleviate this issue?

Answer 23

Telomerase is an enzyme that produces telomeres which act as the eylet of a shoelacd for DNA

Telomeres are non-coding sequences attach to the end of the parental strands 3’ overhang

This allows the parental strand to be elongated and create more base pairs to prevent shortening of the daughter strand

The 3’ end of the telomere allows DNA polymerase to fill the gap from the removal of the RNA primer

Question 24

What is the Hayflick limit? Suppose a cell line experienced a mutation that led to
an extremely hyperactive telomerase enzyme. Would it increase or decrease the Hayflick limit?

Answer 24

The Hayflil limit is the number of cell divisions a cell can undergo before telomers shorten and apoptosis occurs.

Hyperactive telomerase would increase the hayflick limit as it would mitigate shortening of the chromosome with every cell division allowing there to be more cell divisions before cell death occurs.

Question 25

Compare and contrast PCR with DNA replication in vivo.

What are limitations of PCR

Answer 25

PCR requires Taq polymerase opposed to DNA polymerase to withstand high heat conditions required to separate DNA strands and elongate parental strands

PCR uses synthetic DNA primers to make billions of copies of DNA, whereas replication uses RNA primers to elongate single daughter strand in once cycle

PCR denatures DNA to separate strands opposed to using helicase like in DNA Replicaiton

PCR uses heat to keep ssDNA stabilized and separated opposed to ssBP

Limitations:
prior knowledge of target seq needed
limited to 10-15 fragments being amplified

Question 26

In gel electrophoresis, what property of DNA makes it migrate through the gel? Suppose you see
two bands on a gel different distances from the well after migration, which band is the smaller
fragment of DNA?

Answer 26

The negative charge of the phosphate group of DNA allows it to move to the positive cathode of the gel electrophoresis.

The band that is furthest from the wells indicates that it is the smaller fragment as larger fragments move slower

Question 27

Why do ddNTPs stop the elongation of new DNA strands? What are two disadvantages to Sanger sequencing?

Answer 27

ddNTPs stop elongation as there is a hydrogen at the 3’ carbon instead of a 3’ OH preventing a phosphodiester bond from forming with the 5’ Phosphate of the nucleotide in the DNA sequence

Disadvantages of Sanger Sequencing?
Must set of 4 rxns for each ddNTP
high effort
need to have prior knowledge of target seq to design primer

Question 28

How does next-gen sequencing work?

Answer 28

Similar to Sangar sequencing however DNA is annealed complementary to tethered seqs in flow cell

Question 29

Suppose you discover a new species of bird and want to sequence its genome. Would you use Next Generation sequencing or Sanger sequencing? Why?