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Nucleic Acid Biochem I > DNA Replication > Flashcards

DNA Replication Flashcards

Readings: 363 - 402

1
Q

Who is the father of DNA replication research?

A

Arthur Kornberg

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2
Q

Where is DNA replication generally controlled?

A

At the point of initiation on a chromosome or genome

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3
Q

Nucleotide monomers are added in what direction during DNA replication?

A

ADDED in the 5’ to 3’ direction

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4
Q

What are the three proposed models of DNA replication?

A

Conservative, semiconservative and dispersive

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5
Q

How was DNA replication found to be semiconservative?

A

By using 14N and 15N radioactively labeled DNA and fractionating it in a CsCl gradient after bouts of replication.

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6
Q

After first replication, how much DNA is new and old? How about after second replication?

A

50% New and 50% old

75% new and 25% old

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7
Q

What is the direction of DNA synthesis along the ‘template strand?’

A

3’ to 5’ for both lagging and leading strand

Lagging strand defined as one being synthesized away from the fork

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8
Q

After a dNTP (deoxynucleoside 5’-triphosphate) is attached to a growing strand of DNA, what is a by product that comes out (and is not incorporated into the nucleic acid)?

A

A pyrophosphate (two phosphates that are hydrolyzed from the dNTP)

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9
Q

What happens at the molecular level when a incoming deoxynucleoside 5’triphosphate is attached to a growing strand of DNA? What enzyme drives this reaction?

A

The 3’ hydroxyl donates a pair of electrons to the phosphorous of the alpha phosphate group, causing a nucleophilic substitution reaction where pyrophosphate is hydrozyled off and the alpha phosphate is attached to the sugar backbone of the DNA polymer.

Pyrophosphatase drives this reaction

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10
Q

Pyrophosphatase drives what reaction?

A

The hydrolysis of PPi in DNA replication, nucleophilic substitution between a 3’ OH and the alpha phosphate of a deoxynucleoside 5’-triphosphate

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11
Q

Which DNA polymerase exhibits 5’ to 3’ exonuclease activity? (there is only ONE)

A

Polymerase I

This enzyme processes okazaki fragments and repairs DNA

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12
Q

What is the main polymerase in DNA replication?

A

DNA polymerase III

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13
Q

True or false? Polymerase I, II and III all exhibit 3’ to 5’ exonuclease activity.

A

True

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14
Q

What direction does polymerase III exhibit it’s exonuclease activity in?

A

3’ to 5’

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15
Q

What are the three steps of closing a nick in DNA during DNA replication? What two products come out of the final step with DNA ligase?

A
  1. Adenylation of DNA ligase
  2. Activation of 5’ phosphate in nick
  3. Displacement of AMP seals nick

AMP and sealed DNA

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16
Q

What are the three MAIN components of DNA polymerase III?

A
  • Pol III core
  • Clamp loader
  • β sliding clamp
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17
Q

What is needed for DNA Pol III β sliding clamp to attach to DNA? Describe steps

A
  1. The clamp loader is bound to ATP which changes its conformation to accept the β sliding clamp
  2. The β sliding clamp/clamp loader attach to a strand of DNA where a RNA primer is
  3. ATP is hydrolyzed to ADP and the clamp loader detaches
  4. The β sliding clamp is still attached to the DNA and ready to slide along the strand
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18
Q

What are two types of synthesis that DNA polymerase III can do (dependent on the β sliding clamp)?

A

Distributive synthesis - Pol III core with no clamp, must attach and detach continuously to synthesize DNA, not very efficient

Processive synthesis - Pol III core is attached to clamp and slides along the strand. Poly III still comes off strand after synthesizing, but stays attached to clamp, keeping the process more efficient

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19
Q

Describe steps of DNA replication, prescribe an enzyme to each step.

A
  1. Processive synthesis by DNA polymerase starts by clamp and Pol III attaching to DNA strand
  2. DNA is unwound by DNA helicase
  3. Untwisting by Topoisomerase
  4. RNA priming by RNA primase
  5. DNA sealing by DNA ligase
  6. ssDNA protection by single strand binding protein (SSB)
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20
Q

Describe DNA synthesis along lagging strand with enzymes for each step.

A
  1. RNA priming (RNA primase)
  2. Clamp loading (Pol I)
  3. Okazaki fragment maturation and nick sealing (Ligase-Pol I)
  4. Polymerase hpping
21
Q

Describe the steps of okazaki fragment nick sealing

A
  1. Pol III core dissociates from β sliding clamp after completing okazaki fragment
  2. Pol I targets the β sliding clamp to remove the RNA primer
  3. DNA ligase targets the to join the okazaki fragments
  4. The unoccupied β sliding clamp is opened
  5. The clamp is unloaded from DNA by excess δ subunits of the clamp loader
22
Q

What does DNA topoisomerase IV do?

A

Unwinds catenated chromosomes.

23
Q

What does the wound up part of catenated chromosomes sometimes do for gene expression?

A

Acts as a termination region when replication occurs without unwinding

24
Q

What are patterns of high and low activity during DNA replication indications of?

A

Bidirectional replication

25
Q

What does DNA polymerase I do?

A
  • Removes RNA primers from okazaki fragments and attaches dNTPs
  • Can be used to make readioactive
26
Q

What is the difference between exonuclease and endonuclease activity?

A
  • Exonuclease activity cleave nucleotides at the ends of a DNA molecule, while endonuclease cleave in the middle
  • Exonuclease results in nucleotides/nucleosides, endonuclease results in oligonucleotides (four nucleotide residues)
27
Q

What is the only amino acid that has amino group in sidechain? How is this used in DNA replication?

A

Lysine, responsible for DNA ligase activity

28
Q

How does lysine act in DNA ligase to seal nicks?

A
  • Acts with ATP to hydrolyze 2 phosphates (ppi) and form the enzyme-AMP complex
  • This activates 5’ phosphate in nuckand through nucleophilic substitution with 3’ OH the nick is sealed by DNA ligase
29
Q

3’ to 5’ exonuclease activity is mainly for?

A

Proofreading

30
Q

What is processivity?

A

How long DNA pol can travel along DNA before falling off. DNA pol III has a beta clamp which extends this greatly

31
Q

What do single stranded binding proteins prevent?

A

Prevents single strands from tangling up into hairpins

32
Q

What is a structural difference between DNA replication machinery of eukaryotes and prokaryotes?

A

Eukaryotes tend to have molecular machinery with more subunits

33
Q

What does telomerase do?

A

Avoids eventual degradation of genomic information by extending 3’ end and adding telomeric sequence

34
Q

How does telomerase add a telomeric sequence to DNA?

A
  • Telomerase has template RNA molecule that allows overhang to be extended, translocates and repeats this extending.
  • RNA acts as RNA primer after priming by primase and polymerase fills the gap, ligase seals the nick
  • Telomere single stranded proteins protect 3’ end
35
Q

Cells with low rates of division contain low to no levels of what enzyme? Why?

A

Telomerase

To prevent cancer

36
Q

How can mismatched DNA be repaired without the assistance of reparative enzymes?

A

If the mutation occurs in the strand that is not used as a template for replication, it may be corrected in future generations.

37
Q

When can a frameshift mutation occur without consequence?

A

In sequences with many short repeating sequences, a 3 nucleotide frameshift may not have consequences. This also makes it difficult for DNA polymerase to detect an error, triplet expansion diseases can result from this sometimes.

38
Q

Polymerase I degrades RNA primers in what direction?

A

5’ to 3’ exonuclease activity

39
Q

What is nick translation?

A

Where Pol I goes down a DNA strand 5’ to 3’, exonucleased NMPs are released and then correct dNTPs are added. This creates a nick that translates down a strand of DNA

40
Q

What does magnesium ion do for polymerase?

A

Deprotonates the 3’ OH group to form the O- nucleophile that will bind the incoming dNTP and facilitate departure of the pyrophosphate leaving group

41
Q

What is another name for DNA helicase in prokaryotes?

A

DnaB

42
Q

What does DnaA do?

A

When growth is triggered in a cell, DnaA proteins accumulate around OriC (origin sites) at DNA to initiate replication by promoting unwinding at the origin site (creating the replication bubble by weakening A=T bonds)

43
Q

What is another name for primase in prokaryotes?

A

DnaG

44
Q

How does DNA ligase ensure that all RNA at the end of an Okazaki fragment is removed before the nick is sealed?

A

By oly acting of a 5’ terminus of DNA, not on RNA

45
Q

What are the components of a replisome?

A
  • Pol III holoenzyme
  • DnaB helicase
  • Primase
46
Q

What is ‘polymerase hopping?’

A

Polymerase can hop from clamp to clamp, leaving clamps on strands of DNA. These clamps have additional uses, but must be eventually recycled

47
Q

What are AAA+ proteins?

A

These bind and hydrolyze ATP.

DnaA is an AAA+ protein that initiates DNA replication

48
Q

What type of DNA sites control the termination of replication in E. coli?

A

Ter sites, which block replication and cause the daughter strand to become catenated with the parent strand. Specialized type II topoisomerases unlink these catenated daughter chromosomes

49
Q

What is a t-loop?

A

The 3’-terminal single stranded DNAis folded back and hybridized to the duplex to form this single stranded loop. This prevents telomerase from binding and creating abnormally long telomeres.

Nucleic Acid Biochem I (5 decks)

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