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MBB 222 > Lecture 10 > Flashcards

Lecture 10 Flashcards

1
Q

What model does DNA replication follow

A

Semi-conservative model

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2
Q

The substrates for

DNA synthesis are?

A

Nucleoside triphosphates (NTPs)

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3
Q

When each nucleotide is incorporated into the growing DNA strand, what bond forms with what and what bond breaks?

A

When each nucleotide is incorporated into the growing DNA strand, it forms a new phosphodiester bond with the last nucleotide added, and the bond between the first phosphate, Pa, and the second phosphate, Pb, of the NTP breaks.

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4
Q

What is DNA replication

A

duplication of chromosomes for the purpose of cell division

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5
Q

Replication occurs by addition of NMPs to the ___ of a nascent (new) strand

A

3’-OH

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6
Q

The correct NTP substrate is selected based on

A

its base pair complementarity with the nucleotides in the existing strand to be duplicated. This strand acts as a “template”, dictating the nucleotide sequence of the newly-synthesized “daughter” strand

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7
Q

Where do incoming nucleotides get positioned?

A

incoming nucleotide gets positioned adjacent to the 1st non-paired nucleotide on the template strand through Watson-Crick base pairing between the incoming dNTP base and the template base

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8
Q

Where is the nucleophilic attack done on during DNA replication and by which molecule

A

nucleophilic attack by the 3’-OH of the most recently added nucleotide (on the primer/daughter strand) on the alpha-phosphate of the incoming dNTP

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9
Q

Which way does DNA synthesis occur?

A

DNA synthesis always occurs in the 5’’ -> 3’ direction, with dNMPs being added iteratively to the 3’ end of the primer/daughter strand

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10
Q

Why does DNA synthesis occur 5’ -> 3’

A

because of the requirement for the 3’- OH from the primer strand and the 5’ phosphate on the incoming dNTP substrate

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11
Q

The raw materials for DNA replication are (5)

A
  1. a DNA template
  2. deoxynucleoside triphosphates (dNTPs)
  3. a protein complex that includes the enzyme DNA polymerase (DNA is a polymer of nucleotides)
  4. a DNA or RNA ‘primer’ to provide a 3’ OH
  5. Mg2+ ions (cofactor for the polymerase)
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12
Q

How do you unwind DNA to expose the template strand bases?

A

In vitro this is done by heating up the DNA to form two separate template strands. Short DNA oligonucleotides (primers) are added that are complementary to a segment of the template strands. The mixture is cooled and the primers anneal to the template strand. DNA synthesis extends the primer strands in the 5’ ® 3’ direction

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13
Q

the DNA ends are labeled

A

according to the exposed end of the sugar:phosphate backbone.

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14
Q

DNA synthesis in vivo requires an ___ primer synthesized by an ___
polymerase

A
  • RNA primer

- RNA polymerase

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15
Q

What are helicases

A

Enzymes that unwind DNA in vivo to expose the bases on each of the template strands

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16
Q

DNA replication is primed by?

A

a short RNA primer

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17
Q

RNA primer is synthesized by?

A

Primase, an RNA polymerase

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18
Q

DNA polymerases require a pre-existing what

A

DNA or RNA primer and template

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19
Q

How is the RNA primer removed after

A

at a later stage of replication and replaced by DNA

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20
Q

Difference between DNA and RNA polymerase?

A

DNA polymerases require a pre-existing DNA or RNA primer and template. They cannot synthesize DNA de novo. They must add incoming dNMPs to an existing primer with a 3’-OH. (RNA polymerases do not need a primer and can synthesize RNA from scratch.)

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21
Q

Where does DNA synthesis occur?

A

DNA synthesis occurs at replication forks simultaneously for both parent strands, always in the 5’ -> 3’ direction

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22
Q

Which strand is synthesized continuously?

A

Leading strand

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23
Q

Which strand is synthesized in short pieces?

A

Lagging strand

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24
Q

What are Okazaki fragments?

A

short pieces synthesized for the lagging strand

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25
Q

Both new strands are synthesized in a coordinated

fashion by

A

a single dimeric DNA polymerase III complex (DNA pol III)

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26
Q

How are the leading and lagging strands, which run in

opposite directions, both produced in the same direction by the DNA polymerase III complex?

A

The template for the lagging strand loops

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27
Q

What is the origins of replication

A

In all cells (eukaryotic, prokaryotic) with circular or linear DNA molecules, DNA replication begins at defined sequences termed “origins of replication” (ori).

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28
Q

Initiation of replication is controlled by?

A

proteins that recognize and bind to the DNA sequence of the origin of replication

29
Q

Are there usually one or more origins

A

Often there are multiple origins

30
Q

What has circular chromosomes with a single origin of replication - “Ori C”

A

Prokaryoates like E. coli

31
Q

the ori opens up (DNA strands

are separated by a helicase) to form ___ replication forks

A

2 replication forks

32
Q

replication is ____, with a DNA polymerase complex operating at each of the 2 forks

A

bidirectional

33
Q

Prokaryotes vs eukaryotes in terms of ori

A

Prokaryotes have a single origin of replication, eukaryotic genomes
have thousands

34
Q

Why isn’t the human genome replicated from a single origin

A

Replication of the human genome from a single origin with two forks on each chromosome (i.e. bidirectional replication)
would take weeks - need many origins of replication.

35
Q

What are DNA polymerases

A

enzymes that catalyze polynucleotide chain elongation (nucleotide polymerization)

36
Q

What is the role of DNA pol I

A

“proof-reading” and “nick translation” (connecting Okazaki fragments)

aka “clean-up polymerase”
replaces the RNA primer nucleotides (ribonucleotides) with deoxynucleotides in a
process called nick translation
proof-reading functions

37
Q

What is role of DNA pol III

A

major polymerase involved in chain elongation during replication (also has proof-reading function)

main elongation enzyme
highly processive: adds thousands of nucleotides in 5’-3’ direction before falling off the DNA

38
Q

What is an exonuclease (exo)

A

an enzyme that catalyses the hydrolysis of phosphodiester bonds at the end of nucleic acid molecules

39
Q

What direction do exonucleases act ing

A

Some exonucleases act in the 5’  3’direction, others in the 3’  5’ direction

40
Q

What direction does DNA pol I act in

A

DNA pol I has both 5’  3’and 3’  5’exonuclease activity

41
Q

What direction does DNA pol III act in

A

DNA pol III only has 3’  5’exonuclease activity

42
Q

What is primase

A

Primase and helicase complex

43
Q

What is the function of primase

A

Generates RNA primer

44
Q

What is the fxn of helicase

A

Unwinds DNA

45
Q

Fxn of SSBs?

A

Keep DNA unwound

46
Q

Which DNA pol is the “clean up polymerase”

A

DNA pol I

47
Q

Fxn of topoisomerase

A

relieves topological stress generated from unwinding by helicase

48
Q

How many domains do DNA poly I have

A

3

49
Q

What is nick translation?

A

When DNA pol I Removes the RNA primer one ribonucleotide at a time: 5’  3’ exonuclease activity
Adds deoxyribonucleotides to the 3’ end of the adjacent Okazaki fragment (lagging strand): 5’ -> 3’ polymerase activity (polymerization always occurs 5’ -> 3’)

50
Q

What is proof reading

A

Excises mismatched or defective nucleotides in the 3’ end of the Okazaki fragments: 3’ -> 5’ exonuclease

51
Q

Does each okazaki fragment has its own RNA primer?

A

Yes

52
Q

How are okazaki fragments removed

A

DNA pol I simultaneously polymerizes (extends) one end of an Okazaki fragment and excises the RNA primer of an adjacent Okazaki fragment one nucleotide at at time

53
Q

What adds the dNMPs?

A

DNA pol I

54
Q

What happens when the dNMPs are incorrect?

A

DNA pol I excises them (3’  5’ exonuclease activity) and replaces them with the correct nucleotide.

55
Q

Which side are the dNMPs added on the Okazaki fragments?

A

the 3’ end of one Okazaki fragment

56
Q

Which side are ribonucleotides removed from?

A

5’ end of the adjacent RNA primer

57
Q

The nick between two strands get translated along the strand in which direction?

A

5’->3’

58
Q

The nick is sealed by what

A

DNA ligase

59
Q

How many incorrect base pairs are there

A

1 for every 10,000 correct insertions

60
Q

The specificity of replication is dictated by?

A

hydrogen bonding and shape complementarity between the bases on the template strand and the incoming bases as well as by correct fitting of the base pairs in the polymerase active site

61
Q

How can DNA polymerase replicate DNA more faithfully than H bonding interactions between the bases alone can account for

A

some amino acid side chains of the polymerase active site form H bonds with the base pair: “base selection

62
Q

What acts like molecular rulers?

A

H-bonds from bases in the minor groove to amino acid side chains in the active site of polymerase

63
Q

What happens when C tries to pair with A, and G tries to pair with A

A

, if a C tries to pair with A, one of the bases would need to tilt to optimize H-bonding, which would then displace H-bond acceptors in the minor groove. If a G tries to pair with A the geometry would be even worse as both G and A are bicyclic (double-ringed) and would disrupt the spacing between the DNA backbones. Incorrect base pairing would exclude the pair from the active site of the polymerase, allowing it to migrate to the 3’  5’ exonuclease site where the mismatched base is excised.

Phosphodiester bond is either not formed or formed but subsequently excised

64
Q

What is fidelity

A

High accuracy

65
Q

How many subunits is DNA pol consisted of

A

10 types

66
Q

Which subunits form a clamp on each core polymerase complex (DNA pol III)?

A

A pair of B-subunits

67
Q

Function of the B-B-clamps?

A
  • each B-clamps encircles a template/daughter DNA complex like a clamp
  • the B-clamp slides along the DNA and prevents dissociation of the polymerase - dramatically increases the processivity of the polymerase
68
Q

What is processivity

A

the ability of an enzyme to repetitively continue its catalytic function without dissociating from its substrate.

MBB 222 (12 decks)

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