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Molecular Biology Test 3 > Chapter 13 - DNA Replication > Flashcards

Chapter 13 - DNA Replication Flashcards

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1
Q

Initiation

A

1) Parental strands are separated
2) The ssDNA are stabilized
3) A replication bubble is

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2
Q

Elongation

A

1) Assembly of replisome, a multiprotein structure, that undertakes DNA synthesis at the replication fork
2) Replisome moves along the DNA
3) Parental strands unwinds
4) Daughter strands are synthesized.

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3
Q

Termination

A

The joining of the replicons after which the duplicated

chromosomes are separated.

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4
Q

Conditional Lethal

A

Replication mutation. Example: Temperature sensitivity.
Because inability to replicate DNA is fatal, must generate mutants that are able to replicate only under permissive conditions but die otherwise.

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5
Q

Direction of DNA synthesis

A

5’-3’

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6
Q

Precursor for DNA synthesis

A

Nucleoside triphosphate that loses the terminal two phosphate groups in the reaction.

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7
Q

DNA synthesis is required for

A

Semi-conservative DNA replication and DNA-repair

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8
Q

Replicase

A

DNAP III. One bacterial DNA polymerase undertakes semiconservative replication

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9
Q

DNAP α

A

High-fidelity replicase in eukaryotes. Nuclear replication.

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10
Q

DNAP δ

A

High-fidelity replicase in eukaryotes. Lagging strand.

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11
Q

DNAP ε

A

High-fidelity replicase in eukaryotes. Leading strand.

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12
Q

DNAP γ

A

High-fidelity replicase in eukaryotes. Mitochrondrial replication.

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13
Q

E. coli DNA polymerase I

A

Unique 5’ and 3’ exonuclease activity.

A 103 kD polypeptide that can be cleaved into a large subunit (68 kD) and a small subunit (35 kD).

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14
Q

DNAP I Large Subunit

A

Klenow Fragment

  1. Polymerase
  2. 3’-5’ Exonuclease
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15
Q

DNAP I Small Subunit

A

5’-3’ Exonuclease

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16
Q

The fidelity of replication relies on

A

Specificity of base pairing.

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17
Q

Two classes of DNA polymerase-induced errors:

A
  1. Substitutions

2. Frameshifts

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18
Q

Correction of a substitution error

A

Proofreading scrutinizes newly formed base pairs.

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19
Q

Correction of a frameshift error

A

Reduced with greater enzyme processivity.

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20
Q

Processitivity

A

The tendency of the enzyme cycle on and off the DNA strand/template.

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21
Q

Fidelity mechanism for high-fidelity DNAP

A

The geometry of the base-pairing. Improper fitting stalls the DNAP. Moves the DNAP back to remove the mismatched base.

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22
Q

Three domains of many DNAP

A

Form a large cleft that resemble a right hand.
1. The “palm” includes a conserved sequence motifs that
constitute the active site.
2. The “fingers” (variable) involved in positioning the DNA.
3. The “thumb” (variable) binds DNA and important for processivity.

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23
Q

DNAP nuclease activity

A

Resides in a completely different domain from large cleft/hand that positions DNA.

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24
Q

What exposes the template to an incoming nucleotide while held in the DNAP “hand”

A

A sharp turn in the parental strand exposes the template to the incoming nucleotide.
The 3’end to which the nucleotide is being added is anchored by the fingers & palm.

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25
Q

Contacts between DNAP “hand” and DNA

A

Via phosphodiester bond

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26
Q

Rotation of the DNAP during DNA replication

A

The fingers rotate 60-degrees while the thumb rotates 8-degrees towards the palm.
Rotational changes is cyclic.

27
Q

Is the lagging strand DNA replication continuous or discontinuous?

A

The lagging strand is synthesized in sequential fragments (Okazaki fragments) or discontinuously in the 5’-3’ direction and subsequently joined/ligated together.
Hence it is semidiscontinuous replication.

28
Q

What is required to generate and maintain ssDNA for replication?

A
  1. A helicase to separate/unwind the strands of
    DNA using energy provided by hydrolysis of ATP.
  2. A single-stranded binding protein (SSB) is required to
    maintain the separated strands.
29
Q

What do all DNAP require ?

A

A 3’-OH priming end to initiate DNA synthesis or a primer.

30
Q

Methods to provide the free 3’-OH DNAP require to initiate DNA synthesis?

A
  • RNA primer
  • a nick in DNA
  • a priming protein
31
Q

How many times is the RNA primer uses in leading versus lagging strand?

A
  • One (1) time in Leading Strand

- Multiple times in Lagging Strand

32
Q

Primase

A

Synthesizes the RNA chain that provides the priming end

33
Q

What does priming of replication (starting DNA synthesis) on double stranded DNA always require?

A

A replicase (DNA Pol), helicase (DnaB), SSB, and primase (DnaG).

34
Q

E. Coli priming reactions

A
  1. The oriC system - involves the DnaG primase at the fork.
  2. The φX system - requires a primosome to restart
    replication at a damaged fork.
35
Q

How many DNAP catalytic units are required to synthesize the leading and lagging strands?

A

Two dimeric DNAP catalytic units are required:

  • One enzyme unit elongates the leading strand processively in the same direction.
  • The other enzyme unit must move in the reverse direction to synthesize an Okazaki fragment backwards
36
Q

Each monomeric unit of the E. Coli replicase (DNAP III) dimeric structure contains:

A
  • A catalytic core that includes the α, ε, and θ subunits.
  • A dimerization subunit or τ that links 2 catalytic cores.
  • A processivity component that includes the β-ring clamp
    (holds the catalytic core on the template) and γ complex
    clamp loader.
37
Q

Catalytic core includes

A

α, ε, and θ subunits

38
Q

T-unit

A

Dimerization subunit - links 2 catalytic cores.

39
Q

Processivity component

A

Includes the β-ring clamp (holds the catalytic core on the template) and γ complex clamp loader.

40
Q

Clamp loader

A

Uses ATP hydrolysis to open the β-ring/bind β-subunits to a template.
Binding DNA changes the conformation of the β-subunits. Enhances the recruitment of the catalytic core.
Brings the enzyme core to the DNA.
Dimerization component binds the 2 core enzyme.
Each enzyme core synthesizes one of two new strands of DNA.
Clamp loader dissociates (but remains close) once the clamp is positioned on the DNA.
Cyclic process at the lagging strand.

41
Q

How does the β-ring clamp associate with DNA?

A

The β-subunit of DNA Pol III holoenzyme is a head-to-tail dimer. Forms a ring completely around the DNA.
The β-ring clamp “skates” along the DNA. No bonds.
Associates with DNA via the intermediate water molecules
between the clamp and the DNA.

42
Q

Helicase DnaB

A

Responsible for interacting with the primase DnaG to
initiate each Okazaki fragment.
Contacts the τ subunits of the clamp loader to connect the helicase, primase, and the catalytic core.

43
Q

Trombone model

A

Synthesis of the leading strand creates a loop of ssDNA on the lagging strand. The enzyme complex on the lagging strand that “pulls” the ssDNA through the clamp while synthesizing the new strand.

44
Q

Low-fidelity DNAP are also known as

A

Error-prone polymerases.

45
Q

How many subunits do replicases have?

A

Four (4) subunits (tetramers) for catalysis, priming, processivity, and proofreading.

46
Q

DnaB/MCM complex

A

Helicase

47
Q

DnaC/Cdc6

A

Loading helicase/primase

48
Q

SSB/RPA

A

Single-strand maintenance

49
Q

DnaG/DNAPα/primase

A

Priming

50
Q

τ

A

Holoenzyme dimerization

51
Q

Pol I/FEN1

A

RNA removal

52
Q

Ligase/Ligase 1

A

Ligation

53
Q

Pol III core/Pol ε/Pol δ

A

Catalysis

54
Q

Pol ε

A

Elongates the leading strand

55
Q

Pol δ

A

Elongates the lagging strand

56
Q

β/PCNA

A

Processivity

57
Q

Three different DNAP make up eukaryotic replication fork

A

Pol α/primase (priming)
DnaB/MCM (helicase unwinding)
β/PCNA (processivity)

58
Q

Which DNA polymerase expresses a nuclease activity (E. Coli or mammal)?

A

E. coli DNAP I express nuclease activity to remove a primer.

59
Q

How do mammalian DNAP remove a primer?

A
  1. RNase H makes an endonucleolytic cleavage to
    generate a “flap”.
  2. FEN1 is a 5’-3’ exonuclease that recognizes and cleaves the RNA primer “flap”.
60
Q

Two options when replication fork stalls at a damaged base or nick in DNA

A

Lesion bypass OR recombination.

61
Q

Lesion bypass

A

Occurs during replication with an error-prone DNAP on a template that contains a damaged base.
Error-prone DNAP can incorporate a noncomplementary base into the daughter strand.
The mismatch is repaired at a later time.

62
Q

Primosome

A

Required to restart a stalled replication fork after the DNA has been repaired.
Binds and functions to reload a new DnaB to the reinitiate
replication.

63
Q

Recombination (after replication fork stalls)

A

The damaged sequence is excised. The complement of the other daughter duplex crosses over to repair the gap.
Allows replication to continue.

64
Q

Termination of replication in E. coli

A

Called ter site.
~23 bps
Function is unidirectional.
E. coli have 2 cluster of 5 ter sites.
Each set allows the replication fork into the termination region, but does not allow it out the other side = “replication fork trap”.
The linear eukaryotic chromosomes most likely use a modified form of the ligation process.

Molecular Biology Test 3 (5 decks)

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