Lecture 2

Question 1

What are the 3 alternative theories of DNA replication providing an explanation of each?

Answer 1

1) Conservative: 2 parental strands stay together
2) Semiconservative: each daughter strand has 1 parental and 1 new strand
3) Dispersive: DNA is fragmented, both new and old DNA coexist in the same strand

Question 2

What was the experiment performed by Meselson and Stahl (1958) in order to distinguish the 3 hypothesized modes of DNA replication?

Answer 2

1) DNA in E. coli isotopically labeled with 15N: cells grown in media enriched with 15N such that the heavy isotope becomes incorporated into DNA
2) Nitrogen source then switched to 14N such that some cells are labelled with a lighter isotope
3) Used CsCl density gradient ultracentrifugation to separate DNA of different density

Question 3

What were the hypothesized results of the experiment performed by Meselson and Stahl (1958) in order to distinguish the 3 hypothesized modes of DNA replication?

Answer 3

1) Conservative: result in 2 daughter strands 1 HH and 1 LL
2) Semi conservative: DNA replication would result in 2 double stranded DNA molecules each daughter strand having 1 parent heavy and 1 light strand after 1 duplication. If they duplicate again, there will be a proportion of HL + 2LL + LH
3) Dispersive: Each strand is mixed with heavy and light where the amount of heavy is diluted after each generation resulting in a population of lighter and lighter DNA

Question 4

How does Cesium Chloride density gradient ultracentrifugation work to separate DNA of different densities?

Answer 4

Density gradient centrifugating: when CsCl is dissolved in water it dissociates into Cs+ (very massive) and Cl-. So when the solution is spun, the Cs+ ions experience a strong force towards the bottom of the tube so the ions become more dense at the bottom. There is a repulsive force as well that prevents them all from settling on the bottom resulting in a gradient. When DNA is dissolved in the tube, it will migrate to the position that matches its density.

Question 5

What were the OBTAINED results of the experiment performed by Meselson and Stahl (1958) in order to distinguish the 3 hypothesized modes of DNA replication?

Answer 5

Results from CsCl gradient ultracentrifugation consistent with semiconservative replication: Distinct populations of H/H, H/L, and L/L DNA observed!!

Question 6

How does DNA replicate?

Answer 6

DNA replicates semiconservatively: When parental strands separate, each strand serves as a template to make a new complementary strand

Question 7

What is an overview of DNA Replication in 3 steps?

Answer 7

1) Double stranded DNA must unwind
2) When strands come apart, a replication fork is formed where DNA synthesis occurs
3) Each strand serves as a template

Question 8

How are replication forks and bubbles created during DNA replication?

Answer 8

• During replication, DNA strands separate at an origin of replication (ori), making a replication bubble
• At the end of the bubble is a replication fork
• The replication fork moves outward as bubble grows as complementary DNA is simultaneously synthesized by DNA polymerase

Question 9

What did Reiji and Tsuneko Okazaki (1968) show about how DNA is synthesized?

Answer 9

One strand (the leading strand) continuously but synthesizes the other strand (the lagging strand) discontinuously

Question 10

What is DNA replication termed and why?

Answer 10

Termed Semidiscontinuous replication: daughter strand is being synthesized from 5’ to 3’ away from the replication fork on the lagging strand where pieces are discontinuous

Question 11

What are okazaki fragments?

Answer 11

Discontinuous pieces of DNA making up the lagging strand are called Okazaki fragments

Question 12

How are okazaki fragments of the lagging strand linked?

Answer 12

Fragments are then linked by DNA ligase!

Question 13

What did Tsuneko Okazaki (1985) establish, in vivo, about DNA replication?

Answer 13

Tsuneko Okazaki (1985) established that, in vivo, DNA replication was primed by RNA primers

Question 14

What does DNA synthesis require in order to begin?

Answer 14

Furthermore DNA polymerase can only add nucleotides to an existing strand (i.e. a primer). It cannot start from scratch, therefore it needs a primer to polymerize the DNA.

Question 15

What are RNA primers?

Answer 15

RNA primers are short 10 – 12 nucleotide sequences synthesized by the enzyme primase

Question 16

What synthesizes RNA primers for DNA pol?

Answer 16

Primase enzyme

Question 17

What enzyme removes RNA primers on okazaki fragments such that they can be joined?

Answer 17

DNA polymerase I degrades the RNA sequence of the downstream Okazaki fragment, replacing it with newly synthesized DNA (Okazaki fragments have an initial sequence of RNA that needs to be removed and replaced with DNA)

Question 18

What are the 3 major domains of DNA pol I and their functions?

Answer 18

1) DNA polymerase
2) 3’ → 5’ proofreading exonuclease
3) 5’ → 3’ exonuclease

Question 19

What is does DNA pol I do to the nick in DNA during DNA synthesis in 3 steps?

Answer 19

1) At a nick in the DNA, the gap between lagging-strand fragments, Pol I degrades the RNA primer in the 5’~3’ direction, releasing rNMPs, and simultaneously extends the 3’ terminus with dNTPs in the same direction.
2) The net result is movement of the nick in the 5’ → 3’ direction along the DNA until all RNA is removed.
3) DNA ligase can then seal the fragments (not shown here)

Question 20

What is the most commonly found mode of DNA replication in nature?

Answer 20

Bidirectional replication: DNA replication occurs on both sides of the replication fork.

Question 21

What is the experiment and evidence, provided by J. Huberman and A. Tsai, proving that DNA replication is bidirectional?

Answer 21

• Experiment: Tritium (3H) radioisotopically labeled thymidine becomes incorporated into DNA such that DNA is now labelled with low or high amounts of radioactivity
• Autoradiograph Evidence:Dark lines = show DNA synthesized during the high radioactivity impulse is found on either sides of the replication fork. Light lines = show DNA synthesized during low radioactivity impulse found in between

Question 22

What is the θ (theta) mode of replication of circular DNA?

Answer 22

• DNA replication begins at an origin of replication: defined sequence of DNA where machinery for replication is assembled and replication begins
• Bidirectional replication of circular DNA resembles the Greek letter θ

Question 23

Explain the θ replication of E. coli genome?

Answer 23

E. coli has a 4.6 × 106 bp genome on a single circular chromosome with a single origin of replication, from which DNA replication occurs bidirectionally

Question 24

How many origins of replication are there in eukaryotes and prokaryotes?

Answer 24

Most prokaryotes typically have 1 origin of replication while eukaryotes typically have multiple origins of replication

Question 25

What is the enzymology of replication (simplified) in 5 steps?

Answer 25

1) DNA helicase: uses energy from ATP to unwind DNA 2) Topoisomerase: helps relieve the strain that results in the downstream DNA as the DNA is unwound in the replication bubble 3) Single-strand DNA-binding proteins: prevents renaturing of unwound DNA 4) Primase: synthesizes an RNA primer where DNA can be elongated. It associates with helicase where the fork splits called the primosome 5) 2 DNA polymerases synthesizing leading and lagging strands associate in a complex. The template of the lagging strands loops around to allow the polymerase to act discontinuously (forming okazaki fragments) while associating with the polymerase that is synthesizing the leading strand

Question 26

What is the primosome?

Answer 26

Primase associates with helicase where the fork splits called the primosome

Question 27

How can the lagging-strand Pol III synthesize DNA in the opposite direction to replication fork movement, yet remain tethered to the replisome?

Answer 27

To accommodate these opposed directions, the lagging-strand template is pulled up through the polymerase during chain extension to form a loop --> this is denoted as the "trombone model"

Question 28

What is the E. coli DNA polymerase holoenzyme: “trombone model” in 5 steps?

Answer 28

1) Two Pol III cores are attached to beta clamps on the leading and lagging strands, creating a loop. 2) Primase binds to DnaB helicase and synthesizes an RNA primer (purple). 3) The clamp loader assembles a beta clamp onto the new RNA primer. 4) The second lagging-strand Pol III core (third polymerase) assembles with the new clamp while the first lagging-strand Pol III core extends an Okazaki fragment, creating two DNA loops. 5) Polymerase dissociation: The first lagging-strand Pol III core ejects from the beta clamp on the first Okazaki fragment, leaving the clamp on the DNA

Question 29

What are the 5 DNA polymerase(s); DNA polymerase I, II, III, IV, & V in E. coli and their functions?

Answer 29

1) Pol I: DNA repair and replacement/removal of RNA primers. 2) Pol II: DNA repair 3) Pol III: Major DNA replicative enzyme (as shown in the figure) 4) Pol IV & V: DNA repair

Question 30

What does primase do?

Answer 30

synthesize RNA primers

Question 31

What does ligase do?

Answer 31

Stitches Okazaki fragments together

Question 32

What does helicase do?

Answer 32

separates parental strands of DNA, driven by energy from hydrolysis of ATP

Question 33

What do single-strand DNA-binding proteins (SSBs) do?

Answer 33

Stabilizes ssDNA: SSBs prevent re-annealing of DNA strands separated by helicase

Question 34

What do Topoisomerases do?

Answer 34

relieves torsional strain on DNA

Question 35

What are the 2 steps of strand separation and the enzymes involved?

Answer 35

1) Helicase binds and moves along DNA, separating annealed strands as it goes. Helicase hydrolyzes ATP to provide energy required for this process 2) Single-strand DNA-binding protein (SSB) cannot catalyze strand separation, but selectively bind to ssDNA, forming a coat around it, preventing re-annealing

Question 36

What occurs after strand separation and what enzyme is involved?

Answer 36

- As DNA strands are separated, this causes strain downstream of the fork since DNA is a double helix, the strands must rotate around each other when they separate - Supercoiling produces resistance such that DNA polymerase may stall if it’s not removed - To release the tension, a topoisomerase will transiently cut the DNA either in one or both strands, untwist, and then reconnect it. Relieving the strain by changing the DNA topology.

Question 37

What are the 3 characteristics of the highly conserved structure of DNA pol?

Answer 37

1) Cleft between two helices 2) Made of the “palm”, “thumb”, and “finger” domains 3) DNA binding site in the cleft sitting on the (“palm”)

Question 38

What are the purposes of open and closed forms of Pol I?

Answer 38

1) Pol I, open form: Incoming dNTP binds to fingers domain in open form (a) 2) Pol I, closed form: Change in conformation of fingers domain brings dNTP to base-pairing position (b).

Question 39

How does the active site cavity of Pol I accommodate correctly paired bases?

Answer 39

Geometry of the active site in the closed conformation favours correctly paired bases, while incorrectly paired bases are disfavoured

Question 40

What occurs to misincorporated nucleotides?

Answer 40

When an incorrect base is incorporated in the open form despite this being disfavoured, it frays out since it doesn’t correctly base pair with the template. That base is inserted into the 3’ to 5’ exonuclease which cleaves and removes the incorrect base

Question 41

What occurs if a mismatched base is incorporated?

Answer 41

DNA polymerase pauses. Then the incorrect base can be excised by 3’ to 5’ exonuclease allowing polymerase to then continue.

Question 42

What is the error rate of pol III without proof-reading?

Answer 42

about 1-in-100,000

Question 43

What is the error rate of pol III with 3’ to 5’ exonuclease?

Answer 43

about 1-in-10,000,000,000

Question 44

Incorporation of a correct deoxyribonucleotide is favored over an incorrect deoxyribonucleotide. What are the 6 steps that demonstrate this?

Answer 44

1) When an incorrect dNTP enters the insertion site on Pol 1, binding is readily reversed 2) In the rare instance of incorporation of the incorrect dNTP, the process is slow due to the imperfect active-site fit in the closed form. 3) In the favored (rapid) route, the mispaired 3' terminus is shifted to the 3'~5' exonuclease 4) The mismatched nucleotide is excised to re-form the original primed site by 3'~5' exonuclease. 5) This allows Pol I to insert the correct nucleotide on the second try. Binding and incorporation of a correct dNTP is rapid and paves the way for the next round of incorporation. 6) If the incorrect nucleotide remains, the mismatched DNA is slow to act as substrate for the next round of dNTP incorporation

Question 45

Summarize the activities of all 5 DNA pols

Answer 45

1) Pol I, II, III have important exonuclease activities 2) DNA pol I has 5’-3’ exonuclease activity useful for okazaki fragment 3) DNA pol IV and V have no proof reading 5’-3’ exonuclease activity, but these can be used to read through a damaged DNA template to prevent stalling of DNA replication when there’s damage to the template

Question 46

Summarize DNA replication (5)

Answer 46

1) DNA replication is semiconservative: Daughter DNA contains one new strand and one parental strand 2) DNA replication is semidiscontinuous: Leading strand synthesized continuously. Lagging strand synthesized discontinuously as Okazaki fragments 3) Replication of DNA begins at an origin of replication and proceeds through a coordinated process involving multiple proteins and enzymes 4) Most DNA is replicated bidirectionally (but there are exceptions) 5) DNA polymerase favours the incorporation of correct bases and has proof-reading capability (3’→ 5’ exonuclease activity)