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EoM Exam 2 - Sollars > DNA Replication 1 - Prokaryotes > Flashcards

DNA Replication 1 - Prokaryotes Flashcards

1
Q

How does Acyclovir exploit the rules of DNA replication?

A

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).

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2
Q

What is Acyclovir’s mechanism of action?

A

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.

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3
Q

What phosphorylates Acyclovir?

A

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

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4
Q

What is Valacyclovir?

A

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.

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5
Q

What is Valacyclovir’s mechanism of action?

A

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

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6
Q

What is Rule 1 of DNA replication?

A

DNA replication is semi-conservative.

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7
Q

How is DNA replication semi-conservative?

A

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.

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8
Q

What DNA strands are found in daughter cells?

A

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.

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9
Q

What is Rule 2 of DNA replication?

A

Replication begins at an origin and proceeds bidirectionally.

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10
Q

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

A

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.

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11
Q

What are replication forks?

A

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

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12
Q

What is a replication bubble?

A

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.

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13
Q

What is Rule 3 of DNA replication?

A

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

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14
Q

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

A

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

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15
Q

How is DNA synthesis semi-discontinuous?

A

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

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16
Q

How is each new DNA strand initiated?

To what will it be complementary?

A

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.

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17
Q

What enzymes are important to prokaryotic DNA replication?

A

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

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18
Q

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

A

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.

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19
Q

What is the function of DNA Ligase?

A

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

20
Q

What is the function of DNA Polymerases?

What do they require?

A

DNA Polymerases are responsible for strand elongation.

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

21
Q

What is the function of Helicase?

A

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

22
Q

What is the function of Nucleases?

What is the mechanism of action of each?

A

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.

23
Q

What is the function of Primase?

A

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

24
Q

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

A

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.

25
Q

What are the 3 steps of DNA replication?

How was the molecular mechanism of DNA replication determined?

A

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.

26
Q

What is the process of Initiation?

A

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.

27
Q

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

A

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

28
Q

What is the process of Elongation?

A

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)

29
Q

What are RNA primers required in Elongation?

A

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.

30
Q

Which is the leading strand in Elongation?

A

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

31
Q

Which is the lagging strand in Elongation?

A

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

32
Q

How are Okazaki fragments synthesized in Elongation?

A

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.

33
Q

What is DNA Gyrase?

What is the function of DNA Gyrase in Elongation?

A

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.

34
Q

How are RNA primers removed during Elongation?

What is the other function of this enzyme?

A

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.

35
Q

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

A

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

36
Q

What are Topoisomerase II Poisons?

What is their function?

A

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.

37
Q

What are Catalytic Inhibitors?

What are their functions?

A

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)

38
Q

What is Proofreading in Elongation?

A

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

39
Q

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

A

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

40
Q

What is the process of Termination?

A

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.

41
Q

What 3 aspects ensure fidelity of genetic information information?

A
  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.
42
Q

What other function does methylation serve in prokaryotes?

A

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

43
Q

What is the function of prokaryotic DNA polymerase I?

A

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

44
Q

What is the function of prokaryotic DNA polymerase II?

A

DNA polymerase II is involved in DNA repair functions.

45
Q

What is the function of prokaryotic DNA polymerase III?

A

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

46
Q

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

A

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

47
Q

Which DNA polymerase is the main workhorse in prokaryotes?

A

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

EoM Exam 2 - Sollars (5 decks)

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