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CORE Block 2 > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

Why is DNA replication tightly regulated?

A

It is tightly regulated because it must be performed with high fidelity.

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2
Q

How do we know that DNA replicates according to a semiconservative method?

A

Meselson and Stahl performed an experiment in E. coli in which DNA made with N15 (heavy) was let to replicate in N14 (light) followed by another round of replication in N15 (heavy). Density gradient centrifugation revealed the presence of molecules containing both heavy and light chains, supporting that one parent strand is kept by the daughter molecule in DNA replication.

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3
Q

Which DNA strand acts as a template in DNA replication?

A

Both will act as a template for its own daughter strand by DNA polymerase.

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4
Q

Describe the structure and shape of DNA polymerase.

A

The DNA sits in a groove between the palm and thumb of the DNA polymerase enzyme.

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5
Q

Where is the energy derived for the formation of new phosphodiester bonds in DNA replication?

A

From then hydrolysis of ATP – release of pyrophosphate.

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6
Q

Why is DNA replication so tightly regulated?

A

Because it must be performed with high fidelity. It is the storage of genetic information.

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7
Q

What are the substrates of DNA pol?

A

dNTPs

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8
Q

In the formation of a phosphodiester bond, which parts are the electrophile and which are the nucleophile?

A

The 3’ OH of the growing strand is the nucleophile, and the 5’ phosphate of the new nucleotide is the electrophile.

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9
Q

What drives the formation of new DNA strands?

A

The energy released from the release of the pyrophosphate from dNTP.

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10
Q

What is the origin of DNA replication in prokaryotes?

A

oriC (short repeated AT sequence)

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11
Q

What is the initiation of DNA replication called?

A

Origin firing.

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12
Q

Why do eukaryotes have multiple replication bubbles?

A

Because chromosomes are so long, having multiple origins of replication make replication more efficient.

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13
Q

Where does DNA replication end in eukaryotes?

A

At telomeres.

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14
Q

Describe termination of DNA replication in prokaryotes.

A

Replication forks meet in a region containing multiple copies of a short sequence (TER sites) to which the Tus (helicase inhibitor) protein binds and arrests replication fork by inhibiting strand separation (unwinding).

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15
Q

Which complex is the first to assemble at the site of DNA replication initiation?

A

The primosome.

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16
Q

Describe the prokaryotic primosome.

A
  • DnaA: DNA-binding protein that binds the oriC and melts AT-rich sequence. Directs other pimosome proteins to the oriC.
  • DnaB: a DNA helicase that further separates DNA and uses energy from ATP to do so.
  • DnaC: assitsts in the loading of DnaB onto the DNA.
  • SSB: single-stranded binding protein. Stabilizes DNA single strands which will go on to act as templates for replication.
  • DnaG: DNA primase generates short RNA primers for DNA synthesis.
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17
Q

Describe the structure of DNA helicase (DnaB) in prok.

A

It has a 6-subunit ring structure (hexameric).

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18
Q

Describe the activity of DNA helicase.

A

It binds to and moves along the single stranded region of DNA. Once the helicase encounters a region of double-stranded DNA, it uses its ATPase activity to pry the strands apart at replication forks.

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19
Q

Describe the role of ssBP.

A

Single-stranded binding proteins ensure that single stranded DNA does not form tertiary structures such as hairpins when unwound. This would cause progression of DNA pol to be inhibited.

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20
Q

Why do ssBP’s not cover the bases of the DNA they bind to?

A

So that the bases can still be used as a template for DNA pol.

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21
Q

How many RNA primers are found on the leading daughter strand in DNA replication?

A

Only one primer is used.

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22
Q

How does DNA primase differ from DNA pol?

A

It can start a new polynucleotide chain by joining together two ribonucleoside triphosphates. DNA pol can elongate the primer because it has a 3’ hydroxyl group.

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23
Q

How can parental and daughter strands be distinguished in DNA replication?

A

Daughter strands will have primers and parental strands will not.

24
Q

What does it mean that DNA replication occurs in a semicontinuous fashion?

A

It measn that the leading strand is synthesized continuously, but the lagging strand is synthesized in fragments because it must still be synthesized 5’->3’.

25
Q

Which DNA polymerase synthesizes DNA starting at primers?

A

DNA pol III

26
Q

Describe the role and activity of DNA ligase.

A

DNA ligase seals nicks between DNA fragments by forming phosphodiester bonds between 5’ and 3’ ends. This requires the hydrolysis of ATP.

27
Q

Describe the components of the replisome.

A

The replisome is involved in DNA chain elongation:

  • DNA pol III and sliding clamp to allow DNA pol III to move along the daughter strands.
  • clamp loader: allows sliding clamp and DNA pol III to join DNA.
  • ssBP’s: required to maintain single strands of DNA to act as templates.
  • DNA helicase (DnaB) and DNA primase: also part of primosome, needed for Okazaki fragment formation.
28
Q

In prok, which DNA pol is monomeric and which is heteromeric?

A

DNA pol I is monomeric, and DNA pol III is heteromeric.

29
Q

Which DNA pol in prok are involved in DNA repair?

A

DNA pol II, IV, and V.

30
Q

Describe the function and activities of DNA pol I.

A

DNA pol I removes DNA primers and replaces them with DNA. Has a slow processivity. Elongates 5’->3’, can exonucleate (proofreading) 3’->5’, and remove primers with exonuclease 5’->3’.

31
Q

Describe the function and activities of DNA pol III.

A

DNA pol III recognizes RNA primers and elongates DNA in the 5’->3’ direction starting from the 3’ end of the primer. It is the major replicative polymerase and has high processivity. It also has 3’->5’ proofreading exonuclesae.

32
Q

What is processivity mean in the context of DNA replication?

A

It is a measure of how man nucleotides DNA pol can incorporate in a single DNA binding event (i.e. DNA pol is able to fall off the DNA and rebind)

33
Q

How does DNA pol III and the sliding clamp associate?

A

The two halves of the sliding clamp are loaded onto the DNA by the clamp loader. The sliding clamp binds to the back of DNA pol III.

34
Q

What structure helps DNA pol III have high processivity (stay bound to DNA for long amounts of time)?

A

Sliding clamp.

35
Q

How does DNA pol I and III know to switch from the activity of the polymerase domain to the proofreading domain?

A

Adding the wrong nucleotide will change DNA pol conformation so that 3’->5’ exonuclease activity is activated.

36
Q

Which proteins remove RNA primers used in DNA replication in eukaryote?

A

DNA repair enzymes DNA2, Fen1, and RnaseH.

37
Q

Which end of the daughter strand in DNA replication will not be synthesized and lose part of the DNA sequence?

A

The 5’ end of the daughter strand where the primer was placed and later removed.

38
Q

Why are topoisomerases good drug targets?

A

Cancer cell replication or bacterial cell replication can be inhibited. DNA replication will not take place if unwinding of DNA is not stabilized.

39
Q

What is the solution to the torsional stress caused from unwinding DNA during replication?

A

Topoisomerase acts as a reversible nuclease to catalyze breaking and rejoining of DNA to rotate the DNA. It is bound ahead of the replication fork so DNA pol can continue to replicate DNA.

40
Q

Compare Top1 to Top2.

A

Top1 topoisomerase creates a cut in a single DNA strand, and the helix rotates to relieve torsional stress followed by the reformation of the phosphodiester bond as Top1 leaves the DNA. Ahead of the replication fork. Top2 topoisomerase reaction occurs when two DNA double helices become tangled. A double strand break is made, the second helix is passed through the break, and the strands are resealed. Behind the replication fork.

41
Q

Describe the primosome of DNA replication in eukaryotes.

A
  • ORC (origin recognition complex, analagous to DnaA): binds to each origin of replication along the chromosome.
  • MCM complex (analagous to DnaB): bind DNA when ORC is phosphorylated. Also a hexameric ring structure with helicase activity.
  • DNA pol alpha (analagous to DNA primase) is a polymerase and a primase. processive
  • RPA complex (analagous to ssBP)
42
Q

Describe the replisome of DNA replication in eukaryotes.

A
  • RPA complex (ssBP)
  • RFC (replication factor C complex): clamp loader
  • PCNA: sliding clamp
  • DNA pol delta: lagging strand polymerase. processive!
  • DNA pol epsilon: leading strand polymerase. processive!
43
Q

Describe the localization and function of eukaryotic DNA pol alpha.

A

It is localized in the nucleus and is involved in initiation, with primase activity.

44
Q

Describe the localization and function of eukaryotic DNA pol beta.

A

It is localized in the nucleus, and functions in DNA repair.

45
Q

Describe the localization and function of eukaryotic DNA pol delta.

A

It is localized in the nucleus and functions in the elongation of the lagging strand as well as in DNA repair.

46
Q

Describe the localization and function of eukaryotic DNA pol epsilon.

A

It localizes in the nucleus and elongates the leading strand. It also helps with DNA repair.

47
Q

Describe the localization and function of eukaryotic DNA pol gamma.

A

It localizes in the mitochondria and is involved in the replication of mitochondrial DNA.

48
Q

Do nucleosomes assemble ahead or behind the replication fork in eukaryotic DNA replication?

A

They dissemble ahead of the replication fork as replication machinery assembles, and nucleosomes assemble behind the replication fork after replication.

49
Q

When are histones synthesized and when are they added to DNA to form nucleosomes?

A

They are synthesized during S phase and added to newly synthesized DNA.

50
Q

What factors mediate replication-coupled nucleosome assembly?

A

chromatin remodeling/assembly factors and chromatin modifying enzymes. Needed to preserve epigenetic information in different cell types.

51
Q

What are teleomeres?

A

They are non-coding nucleotide sequences at the end of eukaryotic chromosomes that maintain the structural integrity.

52
Q

Describe the sequence of telomeres.

A

It is G-rich.

53
Q

What are T-loops and why do they form at the end of euk chromosomes?

A

They are caused by telomeres coming together in order to hide ends so they are not recognized as DNA breaks. The protein complex shelterin helps with the looping, facilitated by complementary binding.

54
Q

Describe the role of RNA in telomere maintenance.

A

It is a template, not a primer.

55
Q

Describe telomerase.

A

It is a ribonucleoprotein involved in synthesizing the ends of DNA. It contains an RNA molecule that acts as a template for elongating the GC-rich strand of telomeric DNA and as a protein with polymerase activity. It is thought of as a template bearing reverse transcriptase, as it synthesizes DNA from RNA.

56
Q

Describe telomere maintenance.

A

Telomere synthesis occurs from 5’->3’ of the incomplete lagging strand. It binds complementarily via RNA template to the 3’ end of the parent strand, elongating it. DNA polymerase can then fill in the daughter strand, using the elongated parent strand as a template. Telomerase is especially active during development, as genetic information cannot be lost.

57
Q
A

CORE Block 2 (9 decks)

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