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MOLGEN > Ch 11 Lecture (DNA Replication) > Flashcards

Ch 11 Lecture (DNA Replication) Flashcards

1
Q

initiation

A

recognition of the origin of replication by the replicon

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2
Q

elongation

A

replication of the parental duplex by the replisome

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3
Q

replisome

A

assembles at an origin of replication
elongates

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4
Q

joining/termination

A

completion of replication process, includes separation or joining of daughter duplexes

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5
Q

DNA polymerases synthesize DNA in

A

semiconservative replication and DNA repair

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6
Q

All DNA polymerases synthesize

A

5’ to 3’

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7
Q

some repair polymerases function as

A

independent enzymes

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8
Q

replicases are incrporated with other enzymes into a large complex called the

A

replisome

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9
Q

E. coli polymerases

A

Polymerase III is the primary replicase
Polymerase II is required to restart the replication fork after it is stopped by damage
Polymerase I is involved in both error repair and replication
Polymerases IV and V are error-prone polymerases that allow bypass around DNA damage

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10
Q

replicases usually have nuclease activity, meaning they have a

A

3’ to 5’ proofreading function

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11
Q

DNA polmerase I

A

First DNA polymerase to be characterized
Two parts
Klenow fragment
Polymerase and 3’5’ proofreading function
Small fragment
5’3’ exonuclease function
Excises 10 bases at a time

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12
Q

Free base pairing between incoming dNTP and parental strand would allow for

A

mismatches

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13
Q

how do mismatches happen?

A

Twisting of the helix allows for nonstandard pairs such as C=T
Unique four nucleotide combinations called tautomers also allows nonstandard pairs such as C=A

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14
Q

High-fidelity DNA polymerases have a precisely constrained … that favors binding of standard base pairs

A

active site

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15
Q

Rate of incorporation of incorrect nucleotides is …X slower than correct nucleotides

A

10,000

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16
Q

DNA polymerases can also differentiate between

A

rNTPs and dNTPs

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17
Q

A sugar with a 2’-OH cannot be easily accommodated in

A

the nucleotide-binding pocket

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18
Q

processivity

A

The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle
Frameshift fidelity is increased by enzyme processivity, especially in homopolymeric regions

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19
Q

many DNA polymerases have a structure that resembles a

A

right hand

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20
Q

palm

A

primary elements of the polymerase catalytic site

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21
Q

fingers

A

binds to incoming dNTPs and moves the correct dNTP into close contact with the polymerase catalytic site

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22
Q

thumb

A

Maintains the correct position of the 3’-OH and also a strong association between the polymerase and the parental strand to facilitate processivity

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23
Q

metal cations in the palm domain

A

One metal ion reduces the affinity of 3’-OH for its hydrogen
More nucleophilic
Other metal ion stabilizes the negative charges of the β- and gamma-phosphates of the incoming dNTP and the departing pyrophosphate

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23
Q

the palm domain is composed of

A

a β sheet and contains the primary elements of the catalytic site
Also contains two divalent metal cations that interact with the 3’-OH of the primer and the correct paired dNTP

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24
Q

If a newly added nucleotide is mismatched

A

Disruption of nonspecific hydrogen bonding between palm and base pairs in minor groove of new duplex leads to reduced catalysis rate
Fingers cannot rotate towards palm to bind and direct dNTP to active site
The unpaired 3’ region will move into the exonuclease domain

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25
Q

The DNA polymerase complex advances continuously when it synthesizes the

A

leading strand

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26
Q

The lagging strand is synthesized by

A

making short Okazaki fragments that are subsequently joined together

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27
Q

semidiscontinuous replication

A

The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously

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28
Q

Two functions are needed to convert duplex DNA to a single stranded form

A

A helicase is needed to separate the strands of DNA using energy provided by the hydrolysis of ATP
A single-strand DNA binding protein is required to maintain the separated strands

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29
Q

Most helicases are

A

multimeric proteins that initially encircle DNA at a single stranded region next to a duplex region

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30
Q

most helicases have

A

both a double stranded conformation and a single stranded conformation

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31
Q

the movement of helicases results in

A

unwinding of the DNA

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32
Q

How many ATP are hydrolyzed for each base pair unwound by helicase

A

one

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33
Q

Single stranded binding protein in E. coli is a

A

monomer that binds single stranded DNA cooperatively

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34
Q

Once binding has began on a DNA molecule

A

it is rapidly extended along length of ssDNA

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35
Q

Single stranded binding proteins are also needed for

A

repair and recombination mechanisms

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36
Q

All DNA polymerases require a free … to initiate DNA synthesis

A

3’-OH

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37
Q

The free 3’-OH can be provided by

A
  1. RNA primer
  2. Nick in DNA
    Recombination
  3. Preformed RNA (tRNA)
    Retroviruses
  4. Priming protein that provides a nucleotide with a free 3’-OH
    Ser, Thr, Tyr for some linear phages
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38
Q

Only one priming event is required on the

A

leading strand

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39
Q

Only one priming event is required for each

A

Okazaki fragment

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40
Q

DnaG primase

A

DnaG primase is a special RNA polymerase in E. coli
Used only for DNA replication
10 bp primer
Recognizes 3’-GTC-5’ on the parental strand and begins RNA synthesis with pppAG

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41
Q

Multiple core polymerase enzymes are required to

A

synthesize the leading and lagging strands

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42
Q

In E. coli the Active replisome is an

A

asymmetrical trimer with one core polymerase on leading and two on lagging strand

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43
Q

The replisome in E. coli consists of

A

DNA polymerase III holoenzyme, primase, and helicase

44
Q

The holoenzyme contains … catalytic core polymerase subunits

A

three
One on leading strand
Two on lagging strand

45
Q

Composition of DNA polymerase III holoenzyme

A

Three copies of the polymerase III catalytic core
Three copies of the linker protein tau
Three copies of the clamp, each containing
One copy of the clamp loader

46
Q

polymerase III catalytic core contains

A

One alpha subunit for DNA polymerase activity
One epsilon subunit for 3’to5’ proofreading exonuclease
One theta subunit used to stimulate exonuclease activity when mispaired nucleotides are present

47
Q

linker protein tau

A

joins the core enzymes to the sliding clamp loader

48
Q

clamp contains

A

β2 ring that binds the DNA for processivity

49
Q

clamp loader

A

gamma complex of seven proteins that load the clamp on the DNA

50
Q

Assembly of DNA polymerase III

A
  1. The clamp loader hydrolyzes ATP to bind β subunits to a template-primer complex
  2. Binding of β to DNA changes its affinity for DNA polymerase
  3. A core DNA polymerase binds to a clamp
  4. tau trimer binds the core polymerase and stimulates the binding of two core-β2 complexes to form a trimer
  5. DNA polymerase III holoenzyme is formed
51
Q

As DnaB helicase moves along the lagging strand and denatures the parental duplex,

A

SSB coat the single stranded DNA that is produced

51
Q

The clamp loader hydrolyzes ATP to bind β subunits to a template-primer complex

A

Loader binds ATP
β2 clamp binds to loader
Loader-ATP will open clamp and load it onto a template-DNA complex
Binding of DNA stimulates hydrolysis of ATP to ADP
Loader-ADP is released from closed clamp-DNA complex

52
Q

The core on the leading strand displaces the SSB and

A

continues synthesis

53
Q

On the lagging strand, the single stranded DNA that is produced is coated with

A

SSB and looped

54
Q

The size of the loop … as helicase continues to denature the parental duplex

A

increases

55
Q

Simultaneously, one of the lagging strand core polymerases (Lagging Core 1) will be synthesizing the … Okazaki fragment

A

previous

56
Q

At the fork, when the DnaG primase component finds the correct sequence, it will begin

A

primer synthesis

57
Q

Following completion of primer synthesis, the clamp loader will

A

load a new clamp onto the newly primed lagging strand

58
Q

The other lagging strand core (Lagging Core 2) will bind the clamp and begin synthesis of an Okazaki fragment Before

A

synthesis of the previous Okazaki fragment is complete

59
Q

When Lagging Core 1 is finished synthesizing its Okazaki fragment,

A

it will be released from the clamp and disassociate from the lagging strand

60
Q

Interactions between DnaB helicase and tau results in an

A

increase in helicase and core enzyme activity by 10X

61
Q

DnaG primase is the only replisome component that is not

A

tightly associated with the fork

62
Q

The only association between DnaG and the other fork components occurs via

A

weak interactions with DnaB

63
Q

After primer synthesis, DnaG is

A

released back into solution

64
Q

A stronger association between DnaG and DnaB would result in

A

more frequent periods of interaction between the components and more priming events creating shorter Okazaki fragments

65
Q

A weaker association between DnaG and DnaB would result in

A

less frequent interactions and fewer priming events creating longer Okazaki fragments

66
Q

DNA polymerase I

A

removes the primer and replaces it with DNA

67
Q

Mammalian DNA polymerases do not have

A

5’ to 3’ exonuclease activity

68
Q

Okazaki fragment primer removal

A

Synthesis of an Okazaki fragment will displace the primer of the previous fragment as a “flap”
Base of the flap is cleaved by FEN1
DNA polymerase will fill the gap where primer was located
FEN1 also important for preventing hairpins from forming in areas of repeated sequences

69
Q

DNA ligase creates a

A

phosphodiester bond that fills “nicks” in the DNA backbone
Uses an AMP intermediate

70
Q

DNA ligase in replication

A

Connects the terminal 3′-OH produced by DNA Polymerase I to the 5′-P of the first nucleotide previously added by DNA Polymerase III

71
Q

As a replication fork moves along dsDNA, it must

A

unwind and denature the parental duplex

72
Q

For every 10bp replicated

A

one complete turn of the parental duplex is unwound

73
Q

Parental duplex ahead of the fork cannot …, so it will eventually become …

A

freely rotate
overwound

74
Q

Excess positive supercoiling is released via the action of

A

DNA topoisomerase enzymes

75
Q

DNA topoisomerase I

A

No external energy used to create single strand breaks

76
Q

DNA topoisomerase II

A

Uses ATP hydrolysis to create double strand breaks
Bacterial DNA gyrase
Able to disentangle any two duplexes

77
Q

A replication fork stalls when it arrives at

A

damaged DNA
Mispaired nucleotide
DNA break

78
Q

what percent of bacterial forks encounter an error during a replication event

A

18-50%
common occurrence

79
Q

The replication complex must be replaced by a specialized DNA polymerase for

A

lesion bypass

80
Q

error-prone polymerase

A

Polymerase on lesion strand is removed and replaced with an error-prone polymerase
Damage is bypassed
High fidelity polymerase replaces error-prone polymerase

81
Q

The two replication forks usually meet

A

halfway around the bacterial chromosome

82
Q

If replication forks travel beyond their half of the chromosome,

A

ter sites will initiate disassociation of DnaB

83
Q

Ter sites serve as a

A

directional “trap”

84
Q

Eukaryotic replication fork composition

A

One DNA polymerase alpha
One DNA polymerase epsilon
One DNA polymerase delta

85
Q

DNA polymerase alpha

A

initiates DNA synthesis
contains primase domains
binds to the initiation complex
Synthesizes primer plus approximately 20 nucleotides of DNA

86
Q

DNA polymerase epsilon

A

elongation of the leading strand

87
Q

DNA polymerase delta

A

elongation of the lagging strand

88
Q

Leading and lagging strand polymerases act

A

independently of each other

89
Q

Polymerase alpha is replaced by

A

polymerase epsilon/delta on the leading/lagging strands for remaining DNA synthesis

90
Q

Polymerase epsilon is highly processive due to its interaction with

A

CMG helicase (Cdc45-GINS-Mcm)
RFC clamp loader
PCNA processivity clamp

91
Q

The roles of RFC and PCNA are analogous to that of the

A

gamma clamp loader and β2 clamp in prokaryotes

92
Q

On the leading strand

A

CMG helicase moves along the strand and denatures parental duplex
Attached polymerase epsilon will continuously replicate

93
Q

On the lagging strand

A

Polymerase alpha will prime each Okazaki fragment
After polymerase switch, polymerase delta will complete fragment
Eukaryotic Okazaki fragment length of approximately 200 nucleotides

94
Q

There must be some disruption of … during DNA replication

A

nucleosome structure

95
Q

Disruption is confined to

A

the immediate vicinity of replication fork

96
Q

Requires synthesis of enough histone proteins to package entire genome during S phase

A

20 repeated copies of major histone genes in vertebrates
Histone mRNA levels increase 50X in S phase due to
Increased transcription
Reduced degradation
After DNA replication is complete, major histone mRNAs are degraded within minutes

97
Q

Disassembly and reassembly of nucleosomes at replication fork is directly linked to

A

replisome

98
Q

Nucleosome assembly

A

H32 -H42 tetramer binds to DNA, followed by stepwise addition of H2A-H2B dimers
Replication-coupled pathway
Process is facilitated by histone chaperones
Chromatin remodeling complexes
Reverse process for disassembly

99
Q

Nucleosome disassembly occurs at the replication fork

A

H32 -H42 tetramer is released from the duplex but still retained at the fork via direct and indirect associations with the CMG helicase
H2A-H2B dimers are fully released and diffuse away

100
Q

Histone chaperones used in replication-coupled assembly

A

FACT

101
Q

Histone chaperones used in replication-coupled assembly

A

CAF 1
NAP 1

102
Q

CAF1

A

Associated with PCNA clamp
Assembles “old” and “new” H32-H42 onto daughter duplex

103
Q

NAP 1

A

Associated with PCNA clamp
Assembles “old” and “new” H2A-H2B onto daughter duplex

104
Q

FACT

A

Associated with replisome
Has flexible domains that bind to and help disassemble nucleosomes at fork
May also hand H32 -H42 tetramers to replisome components that pass them behind fork to CAF1

105
Q

replication coupled assembly

A
  1. Replication fork displaces octamers
  2. Octamers disassociate into H32-H42 tetramers and H2A-H2B dimers
  3. “Old” H32-H42 tetramers are transferred randomly to one of the two daughter strands by FACT and NAP1
  4. “Old” H2A-H2B dimers are released into the soluble histone pool
  5. “New” H32-H42 tetramers are assembled onto other daughter strand by CAF1
  6. “Old” or “new” H2A-H2B dimer chosen from the soluble pool by NAP1
  7. The H2A-H2B dimer is placed onto daughter strands by NAP1
106
Q

The specific modifications of these tetramers will result in

A

recruitment of specific enzymes that are able to propagate modifications onto the H2A-H2B members of the nucleosome
Also able to propagate modifications onto neighboring nucleosomes

107
Q

Old H32-H42 tetramers (with their associated modifications) are passed to

A

the daughter duplexes

MOLGEN (28 decks)

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