Genetics & Evolutionary Biology > DNA Replication > Flashcards

DNA Replication Flashcards

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1
Q

How is DNA Replication Semi-conservative?

A
  • Each parental strand serves as a template
  • Complementary base pairing determines the sequence of the new strand (ie. hydrogen bonding potential)
  • Each strand of the parental helix is conserved (ie. Double helix is half-conserved → semiconservative)
  • Mechanism of DNA replication was first shown to occur in the E. coli by Meselson and Stahl
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2
Q

Draw and label a diagram of how DNA Replication is Semi-conservative.

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

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3
Q

Which direction of the DNA strand does DNA replication occur?

A
  • There are replication forks at each end of the replication bubble and these forks proceed outward in both directions (ie. Bidirectional replication)
  • Applies to circular DNA
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4
Q

Study the diagram of the Replication in E. coli.

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

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5
Q

What is a Replicon?

A
  • A nucleic acid molecule, or part of one, which replicates as a unit, beginning at a specific site within it
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6
Q

How many points of origin are there in E.coli?

A
  • In bacteria there is usually one unique origin per chromosome, and this single origin controls the replication of the entire chromosome
  • Single origin of replication, called oriC, in E. coli
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7
Q

How many Replicons are there in prokaryotic chromosomes?

A
  • Each origin controls the replication of a unit of DNA called a replicon; thus, most prokaryotic chromosomes contain a single replicon
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8
Q

What sequences are found in the oriC of E. coli?

A
  • Contains two different conserved repeat sequences:
    1. Facilitates the formation of a localized zone of strand separation → replication bubble
    2. Represents binding sites for a protein that plays a key role in the formation of the replication bubble
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9
Q

Provide a summary of How Semi-Conservative DNA Replication works.

A

DNA replicates by a semiconservative mechanism: as the two complementary strands of a parental double helix unwind and separate, each serves as a template for the synthesis of a new complementary strand

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10
Q

What does “nascent” mean?

A
  • (Especially of a process or organization) just coming into existence and beginning to display signs of future potential.
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11
Q

How is the structure of the new DNA strands formed during DNA Replication?

A
  • The hydrogen-bonding potentials of the bases in the template strands specify complementary base sequences in the nascent DNA strands
  • Replication is initiated at unique origins and usually proceeds bidirectionally from each origin
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12
Q

How many hydrogen bonds are there between complimentary base pairs?

A
  • There are two hydrogen bonds between A and T

- There are three hydrogen bonds between G and C

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13
Q

Why are there more T and A nucleotide’s at the origin of Replication?

A
  • The T and A base pairs are easiest to break apart because they only have two hydrogen bonds, hence less energy is needed
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14
Q

What is one of the most important concepts of DNA Replication?

A
  • The sequence or the structure will dictate its function therefore, evolution has dictated that T’s and A’s rather than G’s and C’s are going to be at the point of origin because it’s easier to force the replication bubble to start
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15
Q

What is Helicase?

A
  • Helicases are enzymes that unpack an organism’s genes. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.
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16
Q

What is the process of DNA synthesis assisted by in Vitro?

A
  • DNA synthesis is catalysed by DNA polymerases
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17
Q

What is polymerase?

A
  • An enzyme which brings about the formation of a particular polymer, especially DNA or RNA.
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18
Q

What do DNA polymerases require to catalyse DNA synthesis in vitro?

A

DNA polymerases require:

  • Primer DNA with free 3’-OH
  • Template DNA to specify the sequence of the new strand
  • Substrates: dNTPs (deoxyribonucleotide triphosphates (dTTP, dATP, dCTP, and dGTP))
  • Mg2+
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19
Q

What is Primer DNA?

A
  • A primer is a short single stranded nucleic acid used for the initiation of DNA synthesis.
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20
Q

What does PCR stand for?

A
  • Polymerase Chain Reaction
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21
Q

Draw and label the diagram of DNA synthesis via polymerase.

A

https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing

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22
Q

What does nucleophilic mean?

A
  • (Of a molecule or group) having a tendency to donate electrons or react at electron-poor sites such as protons.
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23
Q

What does DNA Polymerase I do?

A
  • DNA polymerase I catalyses the nucleophilic attack by the 3’-OH at the terminus of the primer strand which is the nucleotidyl phosphorus of the nucleoside triphosphate precursor (dNTP) with the elimination of pyrophosphate (P2O7).
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24
Q

Why is Polymerase I important?

A

The reaction mechanism of polymerase I explains the absolute requirement of DNA polymerase I for a free 3’- OH group on the primer DNA strand that is being covalently extended and dictates that the direction of synthesis is always 5’→ 3’

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25
Draw a diagram of the function of Polymerase I.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
26
What is exonuclease activity?
- Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3' or the 5' end occurs.
27
What is a Oligomer?
- A polymer whose molecules consist of relatively few repeating units
28
How is Exonuclease activity linked to Polymerase I?
- DNA polymerase I contains 5'→ 3' exonuclease activity that cuts back DNA strands starting at 5' termini - The 5'→ 3' exonuclease activity of DNA polymerase I usually excises small oligomers containing up to 10 nucleotides - 3'→ 5' exonuclease activity cleaves off mononucleotides from the 3' termini of DNA strands
29
Study the diagram of how Exonuclease activity works.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
30
Does E. coli have polymerase function?
- Involved in DNA Replication: DNA Polymerases III and I - Involved in DNA Repair: DNA Polymerases II, IV, and V - All of these enzymes synthesize DNA 5' → 3' and require a free 3'-OH at the end of a primer - Not all have exonuclease activity
31
Study the table of polymerase function for E. coli
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
32
What is the proofreading ability of DNA polymerase?
- Proofreading involves scanning the termini of nascent DNA chains for errors and correcting them - This process is carried out by the 3' → 5' exonuclease activities of DNA polymerases
33
How does Proofreading in DNA work?
- When a template-primer DNA has a terminal mismatch, the 3' → 5' exonuclease activity of the DNA polymerase removes the unpaired base or bases - When an appropriately base-paired terminus is produced, the 5' → 3' polymerase activity of the enzyme begins resynthesis by adding nucleotides to the 3' end of the primer strand
34
Give a summary of DNA Synthesis.
- DNA synthesis is catalyzed by enzymes called DNA polymerases - All DNA polymerases require a primer strand, which is extended, and a template strand, which is copied - All DNA polymerases have an absolute requirement for a free 3'-OH on the primer strand, and all DNA synthesis occurs in the 5' to 3' direction - The 3'→5' exonuclease activities of DNA polymerases proofread nascent strands as they are synthesized, removing any mispaired nucleotides at the 3' termini of primer strands
35
During DNA replication, which is the lagging strand and which is the leading strand?
- The strand running in the 5' to 3' direction is called the leading strand while the other strand, with a 3' to 5' sequence, is the lagging strand.
36
Why is DNA in 5' to 3' sequence?
- There are five carbon atoms in the sugar groups, numbered 1' to 5'. The 5' atom has a phosphate group attached to it while the 3' carbon links to an OH group. To form the sides of the helix, the 5' phosphate on one side of the sugar group links to the 3' OH of the next nucleotide. The sequence of this strand is 5' to 3'.
37
How is the Leading strand of DNA replicated?
- The polymerase can only copy DNA in the 5' to 3' direction. This means that it can continuously replicate the leading strand as it moves from the initial point of separation along the strand. - Hence, synthesis of the leading strand is continuous
38
How is the Lagging strand of DNA replicated?
- To copy the lagging strand, the polymerase has to replicate backwards along the strand to the initial point of separation. - Replication then stops, moves up the strand and moves backwards again to the segment that has already been copied. A series of disconnected DNA segment copies called Okazaki fragments are produced from the lagging strand. - Hence, synthesis of the lagging strand is discontinuous
39
What are Okazaki Fragments?
- The new DNA is synthesized in short segments from the lagging strand (Okazaki fragment) that are later joined together
40
What is Ligase?
- An enzyme that joins the disconnected Okazaki Fragments after replication.
41
Study the diagram of Continuous and Discontinuous synthesis of DNA
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
42
What is DNA Primase?
- Primase is an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. Since primase produces RNA molecules, the enzyme is a type of RNA polymerase
43
How many primer strands are needed during replication?
- A single RNA primer is sufficient for the continuous replication of the leading strand, but the discontinuous replication of the lagging strand requires an RNA primer to start the synthesis of each Okazaki fragment
44
What is the primosome?
- A protein complex containing DNA primase and DNA helicase, responsible for creating RNA primers on single stranded DNA during replication.
45
What does the primosome do during replication?
- The initiation of Okazaki fragments on the lagging strand is carried out by the primosome in an ATP-dependent manner
46
In summary what does helicase and primase do?
- DNA helicase unwinds the double helix, and DNA primase synthesizes the RNA primers for successive Okazaki fragments
47
What does polymerase III do?
- RNA primers are covalently extended with deoxyribonucleotides by DNA polymerase III
48
What are DNA topoisomerases
- Enzymes that participate in the overwinding or underwinding of DNA.
49
What does topoisomerase do during replication?
- DNA topoisomerases provide transient breaks in DNA keeping the DNA untangled
50
What does SSB stand for and what is it?
- "Single-Strand DNA-Binding" (SSB) | - SSB protein coats the unwound pre-replicative DNA and keeps it in an extended state for DNA polymerase III
51
What does polymerase I do in the Replication Apparatus?
- The RNA primers are replaced with DNA by polymerase I
52
What does Gyrase do?
- In E. coli, negative super coiling introduced by DNA gyrase (DNA topoisomerase II)
53
In summary, how does replication work at the fork?
- All enzymes and DNA binding proteins function together at each replication fork
54
Draw and label the Replication fork diagram
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
55
What are 5 unique aspects of DNA replication in Eukaryotes?
- Shorter RNA primers and Okazaki fragments - DNA replication only during S phase - Multiple origins of replication - Nucleosomes (prokaryotes don't have) - Telomeres (only at ends of DNA therefore prokaryotes don't have them because they have circular DNA)
56
How many points of origin are in human genomes?
- The genomes of humans contain about 10,000 origins of replication distributed throughout the chromosomes at 30,000 to 300,000-bp intervals
57
In Eukaryotic cells, what determines the points of origin for replication?
- Unclear what factors determine which origins are operational at any given time or in a particular type of cell - Replication initiated at more sites during very rapid cell divisions c/f later stages of development
58
Study the diagram of Bidirectional Replication from multiple Origins in Eukaryotic cells.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
59
What is a Replisome?
- The replisome is a large protein complex that carries out DNA replication, starting at the replication origin
60
Study the diagram of the Eukaryotic Replisome.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
61
What happens to the nucleosomes during replication?
- Nucleosomes appear to have the same structure and spacing immediately behind a replication fork (post -replicative DNA) as they do in front of a replication fork (prereplicative DNA)
62
If nucleosomes are the same during replication, what does this suggest?
- Suggests that nucleosomes must be disassembled to let the replisome duplicate the DNA packaged within them, and then be quickly reassembled - DNA replication and nucleosome assembly must be tightly coupled
63
What is the Telomere problem?
- At the end of the DNA molecule being replicated discontinuously, there would be no DNA strand to provide a free 3'- OH (primer) for polymerization of deoxyribonucleotides after the RNA primer of the terminal Okazaki fragment has been excised
64
What are the possible solutions for the Telomere problem?
- The telomere must have a unique structure that facilitates its replication - There must be a special enzyme that resolves this enigma of replicating the terminus of the lagging strand
65
What is Telomerase?
- A ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres
66
What is the 1st and 2nd steps of the function of Telomerase?
- First the telomerase binds to the Telomere - Second, the Telomerase recognizes the G-rich telomere sequence on the 3' overhang and extends it 5'→3' one repeat unit at a time
67
What is the 3rd step of Telomerase?
- Telomerase does not fill in the gap opposite the 3'-end of the template strand, it extends the 3’-end of the template strand
68
What is the 4th step of Telomerase?
- After several telomere repeat units are added by telomerase, DNA polymerase catalyzes the synthesis of the complementary strand
69
Why is Telomerase important?
- The unique feature of telomerase is that it contains a built-in RNA template - Without telomerase activity, linear chromosomes would become progressively shorter
70
Study the diagram of the process of Telomerase.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
71
How does Telomerase Length link to Cancer and aging?
- Most human somatic cells lack telomerase activity - In many cancer cells genes encoding telomerase are expressed - One approach to cancer treatments → develop telomerase inhibitors - However, other cancer cells do not contain active telomerase - Shorter telomeres are associated with cellular senescence and death - Diseases causing premature aging are associated with short telomeres (eg Progeria)
72
Give a summary on Replication and Telomeres.
- The large DNA molecules in eukaryotic chromosome replicate bidirectionally from multiple origins - Two or three DNA polymerases (α, δ, and/or ε) are present at each replication fork in eukaryotes - Telomeres, the unique sequences at the ends of chromosomes, are added to chromosome by a unique enzyme called telomerase
Genetics & Evolutionary Biology
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