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MOLGEN > Ch 10 Lecture (Replicon) > Flashcards

Ch 10 Lecture (Replicon) Flashcards

1
Q

replicon

A

A unit of the genome in which an individual act of replication occurs
Each contains an origin for initiation of replication

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2
Q

origin of replication

A

Physical site on DNA where DNA is unwound and replication is initiated

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3
Q

replicator

A

Cis-acting DNA sequences sufficient to direct the initiation of DNA replication
Origin is always part of replicator, but is often only a portion of it

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4
Q

terminus

A

A segment of DNA at which replication ends

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5
Q

bacterial chromosomes each contain how many origins of replication

A

one

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6
Q

Eukaryotic chromosomes contain a large number of c

A

unevenly spaced replicons

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7
Q

how many times are replicons fired during eukaryotic replication

A

once

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8
Q

semiconservative replication

A

Both strands of a parental duplex are separated
Each strand then acts as a template for synthesis of a complementary strand

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9
Q

replication fork

A

Point at which replication occurs
Initiated at the origin and then moves sequentially along the parental duplex

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10
Q

replication bubble

A

How a replicated region appears within unreplicated DNA when viewed by electron microscopy
Appearance does not distinguish between unidirectional and bidirectional replication

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11
Q

bacterial chromosomes are usually

A

circles that replicate bidirectionally from a single origin

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12
Q

the early stage of bacterial replication is sometimes called

A

a theta structure

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13
Q

oriC

A

The replication origin of E. coli
245 bp in size

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14
Q

DnaA

A

Bacterial replication initiator protein
Binds to DnaA boxes in oriC

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15
Q

Binding of DnaA to oriC is influenced by

A

interactions with ATP and ADP

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16
Q

OriC contains how many copies of the palindromic sequence GATC

A

11

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17
Q

The adenine in this palindrome is a target for methylation by

A

Dam methylase enzymes

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18
Q

where are dam methylation sites located

A

Dam methylation sites are scattered throughout the genome, but several overlap DnaA boxes

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19
Q

what does Dam do before replication begins

A

the adenines of the palindromic sequence are methylated on both parental strands by Dam

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20
Q

after replication, the DNA is considered

A

hemimethylated

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21
Q

hemimethylated origins

A

inhibit initiation of replication

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22
Q

only fully methylated origins can

A

initiate replication

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23
Q

The GATC sequences at hemimethylated oriC remain

A

hemimethylated following replication much longer than GATC elsewhere in genome

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24
Q

Hemimethylated promoter inhibits expression of

A

DnaA protein
therefore delays re-initiation of replication

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25
Q

Hemimethylation also allows cell to distinguish

A

parental and daughter strands from each other
Cell can determine which strand to use as a template for DNA repair

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26
Q

Three events must occur to initiate replication at oriC

A
  1. Protein synthesis of the DnaA protein
  2. Transcription of one of the two genes that flank oriC
  3. Synthesis of the cell wall
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27
Q

DnaA is the

A

licensing factor that must be made anew in each round of replication

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28
Q

opening of a transcription bubble assists

A

DnaA in opening the replication bubble

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29
Q

drugs that inhibit cell wall formation also inhibit

A

initiation of replication

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30
Q

Initiation at oriC begins with formation of a large six-membered protein complex

A

DnaA, DnaB helicase, DnaC, HU, gyrase, SSB

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31
Q

Only DnaA-ATP will bind a

A

fully methylated oriC

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32
Q

oriC must be associated with

A

the cell membrane for DnaA-ATP binding to occur

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33
Q

DnaA-ATP has an intrinsic ATPase activity that is stimulated by

A

ssDNA and phospholipids

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34
Q

DnaA-ADP is unable to

A

initiate replication

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35
Q

ssDNA is produced by

A

replication bubble formation

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36
Q

Origin of replication remains anchored to the cell membrane for

A

1/3 of the cell cycle

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37
Q

Daughter strands are unable to be

A

methylated while bound to membrane

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38
Q

Opening oriC involves

A

9bp and 13bp repeats within the oriC sequence

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39
Q

The 9bp repeats are the initial binding sites for

A

DnaA-ATP

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40
Q

DnaA contains two distinct

A

DNA binding sites

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41
Q

DnaA-ATP initially binds as

A

an extended multimeric form at the 9 bp repeats

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42
Q

Multimeric extended DnaA-ATP forms a

A

a helical core around which oriC is wound

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43
Q

steps of bacterial replication initiation

A
  1. DnaA-ATP initially binds as an extended multimeric form at the 9 bp repeats
  2. Multimeric extended DnaA-ATP forms a helical core around which oriC is wound
  3. Multimeric extended DnaA-ATP binds to the 13 bp repeats
  4. Multimeric extended DnaA-ATP converts to a compact form that twists open and stabilizes a ssDNA bubble
  5. DnaA then recruits two “pre-priming” complexes
  6. DnaB also catalyzes the loading of DnaG onto the DnaB-(DnaC-ATP) pre-priming complex at each fork
  7. Loading of DnaG onto the pre-priming complex stimulates the dissociation of ATP from DnaC-ATP
  8. DnaB will displace DnaA from oriC as it extends the bubble
  9. Gyrase is also recruited
  10. As helicase opens the bubble, the ssDNA is immediately stabilized by single-strand binding proteins
  11. Protein HU is also found at the replicon
44
Q

Multimeric extended DnaA-ATP converts to a

A

compact form that twists open and stabilizes a ssDNA bubble

45
Q

Transcription of flanking genes assists in

A

opening bubble

46
Q

“pre-priming” complex

A

One hexameric DnaB bound to six DnaC-ATP monomers
One complex per replication fork in bidirectional  replication

47
Q

DnaB is

A

the helicase responsible for unzipping the replication fork along the parental duplex

48
Q

DnaB cannot

A

initially denature dsDNA

49
Q

DnaB can only extend

A

ssDNA that has already been opened

50
Q

DnaC is the

A

DnaB helicase loader

51
Q

DnaC serves as

A

a chaperone to repress DnaB activity until it is needed

52
Q

DnaB also catalyzes the loading of

A

DnaG onto the DnaB-(DnaC-ATP) pre-priming complex at each fork

53
Q

DnaG is the

A

primase required for initiating polymerase action on the leading and lagging strands at each fork

54
Q

Loading of DnaG onto the pre-priming complex stimulates

A

the dissociation of ATP from DnaC-ATP

55
Q

The dissociation of ATP from DnaC-ATP will cause

A

DnaC to be released from the pre-priming complex

56
Q

The release of DnaC from the pre-priming complex will activate

A

DnaB

57
Q

DnaB will displace

A

DnaA from oriC as it extends the bubble

58
Q

what is gyrase

A

type II topoisomerase
Allows one DNA strand to rotate around the other
Reduces positive supercoiling ahead of each fork that would inhibit helicase action

59
Q

As helicase opens the bubble, the ssDNA is immediately stabilized by

A

single strand binding proteins

60
Q

protein HU

A

Stimulates initiation
General DNA binding protein
Thought to bend DNA and catalyze bubble formation

61
Q

How does the cell prevent premature reinitiation od replication

A
  1. OriC is sequestered from Dam methylase
  2. The amount of DnaA available for binding to oriC depends upon competition for its binding to other sites
  3. Influence of Hda protein on ATPase activity of DnaA
62
Q

How is OriC sequestered from Dam methylase?

A

The SeqA protein binds tightly to GATC palindromes in hemimethylated DNA

SeqA protein remains at the origin after initiation and inhibits Dam methylase action

SeqA prevents DnaA from fully binding to oriC and re-initiating replication

SeqA infrequently dissociates from oriC, allowing Dam to methylate new daughter strand

63
Q

what prevents rebinding of SeqA

A

Dam methylase

64
Q

More than … DnaA binding sites available outside of oriC

A

300

65
Q

DnaA acts as

A

transcriptional regulator at the promoter for several genes

66
Q

Control of binding many DnaA molecules at

A

dat locus

67
Q

dat is a

A

a site away from oriC that has a large number of DnaA binding sites

68
Q

Dat locus binds

A

Binds a larger number of DnaA than the origin

69
Q

Hda is recruited to the origin by

A

DNA polymerase

70
Q

Hda enhances

A

ATPase activity of DnaA

71
Q

Exchange of DnaA-ADP for DnaA-ATP is

A

slow

72
Q

replication proceedes at how many bp/min in bacteria and eukaryothes

A

eukaryotes 2000bp/min
bacteria 50kb/min

73
Q

how long are the replicons on a chromosome

A

40-100 kb

74
Q

Individual replicons are activated at characteristic times during

A

S phase

75
Q

If all replicons fired simultaneously, mammalian genomes could be replicated in about

A

60 minutes

76
Q

how long does replication take in somatic cells

A

more than 6 hours

77
Q

Replicons near … are replicated first in the S phase

A

active genes

78
Q

Heterochromatin is replicated … in the S phase

A

late

79
Q

Most eukaryotic replicons do not have … like in bacteria

A

termination sequences

80
Q

Replication forks continue until

A

they meet a fork coming from a neighboring replicon

81
Q

Replication events seem to be localized to

A

foci

82
Q

how many foci are in a nucleus

A

100-300

83
Q

each focus contains how many replication forks

A

more than 300

84
Q

A … is necessary for initiation of replication at each origin in eukaryotes

A

a licensing factor

85
Q

Licensing factor is present in

A

the nucleus prior to replication

86
Q

what happens to the licensing factor during S phase

A

Is removed, inactivated, or destroyed during the S phase

87
Q

the licensing facor is resynthesized in

A

the cytoplasm

88
Q

Initiation of another replication cycle becomes possible only after

A

nuclear membrane breaks down during M phase

89
Q

functional origins in eukaryotes requires

A

relaxed chromatin

90
Q

what is required in yeast for replication

A

Binding site for the origin replication complex (ORC)
A region of DNA with a high AT content
A binding site for proteins that adjust local chromatin structure and facilitate ORC binding

91
Q

prereplicative complex

A

Complex of four components that must form in an ordered manner at each replicator in order to initiate replication

92
Q

what is in the pre-rc

A

ORC, Cdc6, Cdt1, Mcm

93
Q

ORC is … in all eukaryotes

A

highly concerved

94
Q

when can ORC bind to origins

A

anytime

95
Q

when in ORC activated

A

when a part of the pre-RC

96
Q

prereplicative complex steps

A
  1. Binding of the origin recognition complex to an origin
  2. Cdc6 binds to the replicator only in late M phase and early G1
  3. Active Cdt1 is only present in G1
  4. Cdt1 escorts Mcm to the origin
  5. The pre-RC has now been formed
  6. Cdc6 and Cdt1 are released
  7. Mcm is fully loaded onto the origin
97
Q

Cdc6 is deactivated and degraded as S-Cdk levels … in S phase

A

increase

98
Q

what protein inactivates free Cdt1

A

geminin

99
Q

Geminin is specifically degraded in

A

early G1

100
Q

Mcm2-7 contains the eukaryotic

A

helicase

101
Q

Activation of the helicases requires

A

Cdks

102
Q

In the G1 phase, S-Cdk levels are low, so helicase loading is … and activation of helicase is…

A

allowed
not allowed

103
Q

In the S, G2, and early M phases, S-Cdk levels are high, so helicase activation is…
Helicase loading is…

A

allowed
not allowed

104
Q

Once helicases move beyond the origin, they must be … to initiate another replication event

A

reloaded

105
Q

Cell must wait until the next … to initiate more replication events at origins

A

G1 phase

106
Q

S-Cdk levels rise to their maxima in the

A

S phase

107
Q

what happens when SCDK levels are at their maxima

A

Cdc45 and GINS bind Mcm

Mcm and ORC are phosphorylated

The helicase is activated

ORC is inactivated

The Cdc45-GINS-Mcm complex recruit additional proteins that form the active replicative helicase

The CMG helicases are loaded onto single stranded DNA and move away from the origin

ORC is displaced

Any inactive origins with bound ORC that are passed by the replication fork will be inactivated

MOLGEN (28 decks)

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