DNA Replication 1 - Prokaryotes

Question 1

How does Acyclovir exploit the rules of DNA replication?

Answer 1

Viruses like herpes can encode their own DNA polymerases (which are different from eukaryotic host), thus allowing specific targeting by inhibitors like Acyclovir (developed by Gertrude Elion).

Question 2

What is Acyclovir’s mechanism of action?

Answer 2

Fully phosphorylated Acyclovir has a 200-fold greater affinity for the viral DNA polymerase than the cellular version. The chemical structure of Acyclovir is guanine attached to an incomplete ribose ring lacking a 3’OH group necessary for polymerization, thus acting as a chain terminator.

Question 3

What phosphorylates Acyclovir?

Answer 3

Viral encoded thymidine kinase, ensuring phosphorylation mainly occurs in virally infected cells.

Question 4

What is Valacyclovir?

Answer 4

Valacyclovir is a pro-drug, an esterified version of Acyclovir that has greater oral bioavailability (about 55%) than Acyclovir (10-20%). It is available in generic since November 2009.

Question 5

What is Valacyclovir’s mechanism of action?

Answer 5

It is converted by esterases to the active drug Acyclovir as well as the amino acid Valine via hepatic first pass metabolism.

Question 6

What is Rule 1 of DNA replication?

Answer 6

DNA replication is semi-conservative.

Question 7

How is DNA replication semi-conservative?

Answer 7

Genetic information is transmitted from parent to progeny by replication of parental DNA, a process in which two daughter DNA molecules are produced that are identical to the parental DNA molecule. During DNA replication, the two complementary strands of parental DNA are pulled apart and each is then used as a template for the synthesis of a new complementary strand.

Question 8

What DNA strands are found in daughter cells?

Answer 8

During cell division, each daughter cell receives one of the two parental strands so that each daughter cell ends up with one parental strand and one newly synthesized strand.

Question 9

What is Rule 2 of DNA replication?

Answer 9

Replication begins at an origin and proceeds bidirectionally.

Question 10

What are origins of replication? What is usually found in them?

Answer 10

Origins of replication are unique sites at which replication begins. AT base pairs are usually found in them to facilitate local melting of the duplex to ssDNA for replication. (Remember: AT only has 2 H bonds so easier to separate.

Question 11

What are replication forks?

Answer 11

As the two strands unwind and separate, they form the V-shaped fork structures where active synthesis of the DNA polymer occurs.

Question 12

What is a replication bubble?

Answer 12

Replication forks move away from the origin in both directions (bidirectional) to form a replication bubble structure that looks like a small circle (bubble) within the larger circular genome of the prokaryote.

Question 13

What is Rule 3 of DNA replication?

Answer 13

DNA synthesis proceeds in the 5’ –> 3’ direction and is semi-discontinuous.

Question 14

How does DNA synthesis proceed in the 5’ —> 3’ direction?

Answer 14

Deoxynucleotides are added one at a time to the 3’ end of each growing chain.

Question 15

How is DNA synthesis semi-discontinuous?

Answer 15

At each replication fork, synthesis of the leading strand is continuous, while synthesis of the lagging strand is discontinuous.

Question 16

How is each new DNA strand initiated?

To what will it be complementary?

Answer 16

Each new strand is initiated by synthesis of an RNA primer, which is later removed.

The newly synthesized strand will be complementary to the parent (template) strand.

Question 17

What enzymes are important to prokaryotic DNA replication?

Answer 17

AAA+ ATPase (DnaA), DNA Ligase, DNA Polymerases, Helicases, Nucleases, Primase, Topoisomerase

Question 18

What is the function of AAA+ ATPase (DnaA)?

Answer 18

DnaA binds to the origins of replication and dissociates the helical strands. The energy of ATP cleavage is used to produce a conformational change in the DnaA, which forces the strands apart.

Question 19

What is the function of DNA Ligase?

Answer 19

DNA Ligase creates phosphodiester bonds by using the energy of ATP cleavage. This seals nicks in the DNA strand.

Question 20

What is the function of DNA Polymerases?

What do they require?

Answer 20

DNA Polymerases are responsible for strand elongation.

They require an ssDNA as a template and an RNA Primer.

Question 21

What is the function of Helicase?

Answer 21

Helicases cause dissociation of the two strands of the DNA double helix, unwinding the structure using energy released from ATP cleavage.

Question 22

What is the function of Nucleases?

What is the mechanism of action of each?

Answer 22

Nucleases sever phosphodiester bonds of the DNA backbone.

Endonuclease recognizes sequences within the strand and creates a nick there. Exonuclease “chew on the ends” of strands.

Question 23

What is the function of Primase?

Answer 23

Primase is responsible for synthesizing short stretches of RNA complementary to the template DNA strand that serve as a primer for DNA Polymerase.

Question 24

What is the function of Topoisomerases?
What do they both contain?
What is the mechanism of action of each?

Answer 24

Topoisomerases adjust the supercoiling of DNA double helices, both alleviating supercoiling stress and introducing negative supercoiling.
They contain both Endonuclease function and Ligase function.
Type I Topoisomerases cleave one strand of the double helix. Type II Topoisomerases cleave both strands of the double helix.

Question 25

What are the 3 steps of DNA replication? | How was the molecular mechanism of DNA replication determined?

Answer 25

The 3 steps of DNA replication are Initiation, Elongation, and Termination. The molecular mechanism of DNA replication was determined using in vitro modeling of these reactions with E. coli proteins.

Question 26

What is the process of Initiation?

Answer 26

The base sequence at the origin of replication is recognized and bound by the DnaA protein. The two parental strands are pulled apart to form a replication bubble. Helicase uses energy from ATP to break the H bonds holding the base pairs together. This allows the two parental strands of DNA to begin unwinding and forms two replication forks. SSB binds to the single stranded portion of each DNA strand, preventing the strands from re-annealing and protecting them from degradation.

Question 27

What is the function of Single-stranded DNA binding protein (SSB)?

Answer 27

SSB binds to the single stranded portion of each DNA strand to prevent the strands from re-annealing and to protect them from degradation.

Question 28

What is the process of Elongation?

Answer 28

There is a leading and a lagging strand for each of the two replication forks on the chromosome. On each strand, Primase synthesizes a short (10 nucleotides) RNA primer in the 5' --> 3' direction, beginning at the origin on each parental template strand. DNA polymerase III begins synthesizing DNA in the 5' --> 3' direction, beginning at the 3' end of each RNA primer. The newly synthesized strand is complementary and anti-parallel to the the template parental strand. (See page 15 in Notes)

Question 29

What are RNA primers required in Elongation?

Answer 29

RNA primers are required because DNA polymerases are unable to initiate synthesis of DNA and can only extend a strand from the 3' end of a preformed primer.

Question 30

Which is the leading strand in Elongation?

Answer 30

The strand that can be made continuously is the leading strand.

Question 31

Which is the lagging strand in Elongation?

Answer 31

The lagging strand is synthesized discontinuously as a series of small fragments (1000 nucleotides) known as Okazaki fragments.

Question 32

How are Okazaki fragments synthesized in Elongation?

Answer 32

Each Okazaki fragment is initiated by the Primase synthesis of an RNA primer and then completed by the synthesis of DNA by DNA Polymerase III. Each fragment is made in the 5' --> 3' direction. DNA Ligase then seals the nicks between Okazaki fragments, converting them to a continuous strand of DNA.

Question 33

What is DNA Gyrase? | What is the function of DNA Gyrase in Elongation?

Answer 33

DNA Gyrase is a Type II DNA Topoisomerase that provides a "swivel" in front of each replication fork. As the replication fork advances and separates the two parental strands, positive supercoils are formed ahead of the fork because the twists in the double helix become too tight. DNA Gyrase inserts negative supercoils by nicking both strands of DNA, passing them through the nick, then resealing them.

Question 34

How are RNA primers removed during Elongation? | What is the other function of this enzyme?

Answer 34

DNA polymerase I removes the ribonucleotides one at a time from the 5' end of the primer (5' --> 3' Exonuclease). DNA polymerase I also fills in the resulting gaps by synthesizing DNA, beginning at the 3' end of the neighboring Okazaki fragment.

Question 35

Why is the alleviation of DNA supercoiling by Topoisomerases during Elongation used as a drug target?

Answer 35

Drugs that target Type II Topoisomerases are able to interfere with at least one step of the catalytic cycle.

Question 36

What are Topoisomerase II Poisons? | What is their function?

Answer 36

Topoisomerase II Poisons are drugs that stabilize the covalent DNA Topoisomerase II complex (also called the cleavable complex). Topoisomerase II Poisons are used for anti-tumor activities.

Question 37

What are Catalytic Inhibitors? | What are their functions?

Answer 37

Catalytic Inhibitors are drugs that act on any other steps in the catalytic cycle. Catalytic Inhibitors are used for their anti-neoplastic activity (Aclarubican), cardio-protective activity (ICRF-187), or modulator activity to increase the efficacy of other agents (Suramin and Novobiocin). (See page 16 in Notes)

Question 38

What is Proofreading in Elongation?

Answer 38

Both DNA polymerase I and DNA polymerase III have the ability to proofread their work by means of 3' --> 5' Exonuclease activity.

Question 39

What happens if DNA polymerase I or DNA polymerase III makes a mistake during Elongation?

Answer 39

After undergoing 3' --> 5' Exonuclease activity, the resulting unpaired base at the 3' end of the growing strand is removed before synthesis continues.

Question 40

What is the process of Termination?

Answer 40

Replication is completed when the two replication forks meet each other on the side of the circle opposite the origin. There are specific sequences involved as well as DNA Gyrase.

Question 41

What 3 aspects ensure fidelity of genetic information information?

Answer 41

1. Geometry of active site of DNA polymerases only accepts AT and GC base pairs (only room for 1 purine and 1 pyrimidine joined by H-bond). 2. A 3' --> 5' Exonuclease allows for proofreading. 3. Mismatch repair has a window of opportunity based on the DNA methylation of of the template strand to recognize mistakes in the newly synthesized strand.

Question 42

What other function does methylation serve in prokaryotes?

Answer 42

Methylation is also a defense mechanism for prokaryotes, because it allows them to recognize foreign invaders.

Question 43

What is the function of prokaryotic DNA polymerase I?

Answer 43

DNA polymerase I is involved in DNA repair (proofreads 3' --> 5') and removes RNA primers during replication (unique 5' --> 3' Exonuclease activity).

Question 44

What is the function of prokaryotic DNA polymerase II?

Answer 44

DNA polymerase II is involved in DNA repair functions.

Question 45

What is the function of prokaryotic DNA polymerase III?

Answer 45

DNA polymerase III synthesizes most of the DNA on both the leading and lagging strands. It also proofreads 3' --> 5'.

Question 46

What is the function of prokaryotic DNA polymerases IV and V?

Answer 46

DNA polymerases IV and V are involved in DNA repair functions.

Question 47

Which DNA polymerase is the main workhorse in prokaryotes?

Answer 47

DNA polymerase III is the main workhorse in prokaryotes, because it stays in place one it attaches. (DNA polymerase I tends to fall off.)