Lecture 5 and 6 - DNA Replication

Question 1

What was Arthur Kornberg’s goal?

Answer 1

Worked with E. coli cells - Goal was to study replication in vitro - in a test tube

Question 2

What was step one for Arthur Kornberg

Answer 2

Develop an in vitro assay for DNA synthesis
- Grind up E. coli cells to obtain a cell extract that contains all cellular proteins (including the unknown enzyme that makes DNA)
- Add radioactive nucleotide,14C-Thymine deoxynucleotide (14C-dTTP), and all 4 dNTPSs and DNA
- Incubate for a few hours to allow DNA synthesis to occur
- Add percholric acid to stop the reaction and precipitate the DNA and saw that some of the radioactivity went into the insoluble part because the nucleotide got incorporated into the DNA
- Add DNAase enzyme to break down DNA so that the small amount of 14C-dTTP is found in the soluble fraction

Question 3

What was step two for Arthur Kornberg

Answer 3

Purify the DNA synthesizing “activity” from the crude protein fraction
- Involves separating crude protein into “fractions” by size and testing each fraction for DNA synthesizing ability
- The obtained fraction with activity in the assay is called pure DNA polymerase and it can be used to study the protein components of DNA polymerase and to study the mechanism of DNA replication
- Pyrophosphate released during synthesis

Question 4

What were the potential models for how DNA Synthesis proceeded and which actually happens?

Answer 4

1) Random nucleotide addition
2) 5’ to 3’ synthesis
3) 3’ to 5’ synthesis

Question 5

What is Kornberg’s evidence that DNA synthesis is 5’ to 3’

Answer 5

1) Start DNA synthesis in presence of “cold” nucleotides
2) Short pulse of radioactive nucleotide to label only most recently added nucleotides
3a) Add 3’ to 5’ exonuclease = radioactivity lost from DNA (becomes acid soluble - not incorporated into DNA)
3b) Add 5’ to 3’ exonuclease = radioactivity is retained on DNA (becomes acid insoluble - incorporated into DNA)
* Therefore, nucleotides are added to the 3’ end of a DNA molecule

Question 6

What is Kornberg’s evidence that DNA synthesis involves the release of pyrophosphate from deoxynucleotide triphosphate (dNTPs)?

Answer 6

Experiment: template + primer + 4dNTPs + 14C-dATP-32P
Result: Release of 32P pyrophosphate at equal molarity to the amount of 14C-nucleoside incorporated into DNA
* Labelled 3 phosphates from deoxyribose out as alpha, beta, and gamma, and determined the last two phosphates got released

Question 7

What underlies DNA replication and DNA repair?

Answer 7

Base-pairing

Question 8

Bacteria replication bubble creates what?

Answer 8

Replication forks

Question 9

What direction does a continuous replication fork move in?

Answer 9

3’ to 5’

Question 10

What direction does a discontinuous replication fork move in?

Answer 10

5’ to 3’

Question 11

Is the DNA replication fork symmetrical or asymmetrical?

Answer 11

Asymmetrical

Question 12

How is the leading strand synthesized?

Answer 12

5’ to 3’ synthesis in the direction of fork movement

Question 13

Who studied how the lagging strand was synthesized and when?

Answer 13

Reiji and Tsuneko Okazaki - 1968

Question 14

What experiment was carried out to determine how the lagging strand was synthesized?

Answer 14

1) Brief pulse of H3-thymidine to label DNA
2) Denature dsDNA into ssDNA using alkali
3) Separate DNA according to size using a sucrose gradient
4) Measure radioactivity in each fraction

Question 15

What were the possibilities for lagging strand synthesis?

Answer 15

1) Continuous - One peak because the pieces would get larger over time
2) Discontinuous - Two peaks because the pieces were small

Question 16

How are the Okazaki fragments linked together?

Answer 16

DNA ligase is an enzyme that ligates the 5’ and 3’ ends together

Question 17

What experiment was carried out to determine how Okazaki fragments were linked together?

Answer 17

DNA ligase, which is essential for E. coli viability was not ligating the ends when in temperature-sensitive mutants

Question 18

Since DNA ligase is necessary, what would the peaks look like when adding pulses of a radioactive nucleotide?

Answer 18

The first peak would keep rising as the length of the pulse increases since the number of small fragments would continue to increase
* Would still see the incorporation of larger pieces because of the leading strand being synthesized as well without the need for DNA ligase

Question 19

What did the experiments of Okazaki conclude?

Answer 19

1) The newly synthesized DNA on the lagging strand occurs in short discontinuous fragments that soon become linked to older DNA - Okazaki fragments
2) The short DNA fragments are linked by DNA ligase
3) The lagging strand is primed by a short RNA molecule

Question 20

What does the enzyme DNA primase do?

Answer 20

Synthesizes a short RNA primer with a 3’-OH on the lagging strand by bringing in RNA nucleotides so that DNA polymerase can extend it

Question 21

How do we know that short DNA fragments also contain RNA?

Answer 21

Add RNAse while adding the pulse of H3-Uridine to destroy the RNA primer and notice that there are no larger peaks because the leading strand doesn’t use RNA - only Okazaki fragments detected

Question 22

What are the major components of DNA replication machinery?

Answer 22

DNA helicase (Mcm); Single-stranded DNA binding protein (RPA); Sliding clamp (PCNA); DNA polymerase

Question 23

What does helicase do?

Answer 23

Opens up the DNA double helix in front of the replication fork to separate the strands by forming a ring around one of the DNA strands

Question 24

What does the single-stranded DNA binding protein do?

Answer 24

Prevents the DNA strands from folding in on themselves due to complementary base pairing

Question 25

What does the sliding clamp do?

Answer 25

Stabilizes DNA to that DNA polymerase can continue synthesizing by forming a donut around the DNA

Question 26

Explain the PCNA cycle.

Answer 26

1) Sliding clamp opens once it binds to the clamp loader
2) Binds the 3’ end of DNA by ATP hydrolysis and clamp loader falls off so the clamp can close around the DNA
3) Once DNA polymerase has reached the 5’ end of a previous RNA primer on the lagging strand, PCNA falls off

Question 27

What are the DNA replication mechanisms?

Answer 27

• The proteins at a replication fork cooperate to form a replication machine
• The replication fork contains a large complex including polymerases for the leading and lagging strand
• Lagging strand is looped around so that its polymerase can associate with the leading strand polymerase

Question 28

What mechanisms ensure the accuracy of DNA replication?

Answer 28

1) Base pairing precedes covalent attachment of a nucleotide
2) 3’ to 5’ exonuclease activity
3) Mismatch repair

Question 29

How does 3’ to 5’ exonuclease activity of DNA polymerase work?

Answer 29

Proofreading is used and an unpaired 3’-OH end of primer blocks DNA polymerase from elongation until after the 3’-to-5’ exonuclease activity attached to DNA polymerase chews back to create a base-paired 3’-OH end on the primer strand

Question 30

Where does exonuclease activity reside?

Answer 30

In a distinct domain or subunit of DNA polymerase

Question 31

Where was the exonuclease function first identified?

Answer 31

In bacteriophage T7 - mutations in DNA polymerase gene that produce a “mutator” phenotype

Question 32

How does the mismatch repair pathway work?

Answer 32

1) MSH2 and MLH1 protein complex scans newly synthesized DNA
2) Detects mismatches
3) Makes nicks in “new” DNA strand on either side of the mismatch
4) Strand removed and repaired by DNA polymerase I

Question 33

Which strand is more efficient at being repaired and why?

Answer 33

Lagging strand because the nicks from the Okazaki fragments have not yet been ligated

Question 34

What is more likely to occur for people with mutations in the genes involved in DNA mismatch repair?

Answer 34

Very high cancer incidence

Question 35

What do typical prokaryotic replication origins contain?

Answer 35

A single well-characterized replication origin

Question 36

What do eukaryotic replication origins contain?

Answer 36

Yeast - Multiple well-characterized specific sequences
Most eukaryotes - Multiple not well-defined origins

Question 37

How does DNA synthesis get carried out at replication origins?

Answer 37

1) Local opening of DNA helix at the replication origin
2) RNA primer synthesis
3) Leading-strand DNA synthesis begins
4) RNA primers start leading-strand synthesis

Question 38

When does DNA replication take place in eukaryotes?

Answer 38

S phase of the cell cycle

Question 39

How does the regulation of DNA synthesis get carried out?

Answer 39

A large multisubunit complex called the origin recognition complex (ORC) binds to eukaryotic origins of replication and recruits other proteins to origins in G1 of the cell cycle to form the pre-replication complex (pre-RC) which becomes activated and triggers DNA polymerase and helicase activity for the S phase

Question 40

How does DNA synthesis on the lagging strand get completed?

Answer 40

The ends of the chromosomes, telomeres, contain repeated sequences that are protected by a protein complex, sheltrin, and attract a riboprotein, telomerase, to extend the 3’ end until multiple copies are made and it falls off, allowing DNA primase and DNA polymerase to extend the dsDNA

Question 41

When are DNA-dependent DNA polymerases used?

Answer 41

DNA replication

Question 42

When are RNA-dependent DNA polymerases used?

Answer 42

Telomerase, Reverse transcriptase from retroviruses

Question 43

RNA-dependent DNA polymerase

Answer 43

Reverse transcriptase source is from a virus and the RNA genome and reverse transcriptase brought into a cell can make a DNA copy of their genome

Question 44

When are DNA-dependent RNA polymerases used?

Answer 44

Transcription

Question 45

When are RNA-dependent RNA polymerases used?

Answer 45

Influenza, SARS-CoV-2