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Molecular Genetics > Proteins of DNA Replication > Flashcards

Proteins of DNA Replication Flashcards

1
Q

topoisomerase

A

tension is relaxed in the supercoiled genome by this protein

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2
Q

what does initiation involve?

A

initiation involves the origin by DnaA protein

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3
Q

what must occur at the end of replication?

A

termination and separation

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4
Q

DnaA

A
  • melts the oriC
  • binds to short repeated sequences
  • forms an oligomeric complex that melts DNA
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5
Q

DnaC

A
  • chaperone for DnaB
  • six DnaC monomers bind each hexamer of DnaB
  • this complex binds to the origin
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6
Q

DnaB

A
  • hexamer propels the replication fork
  • gyrase, SSB, and HU proteins are also involved
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7
Q

how does DnaA prevent reinitiation at the origin until the next cell cycle?

A
  • DnaA must be bound to ATP in order for the protein to bind the DnaA boxes at the origin
  • after binding oriC, DnaA has ATPase activity, which prevents further binding
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8
Q

what is ATPase activity stimulated by?

A
  • phospholipids of membrane
  • single-stranded DNA
  • stays at origin/membrane at least 1/3 of cell cycle
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9
Q

of the six proteins that bind to oriC, which is unique to the origin, cannot rebind, and is therefore analogous to the eukaryotic licensing factor?

A

DnaA

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10
Q

of the six proteins that bind to oriC, which one has helicase activity and precedes the replisome at the replication fork during the course of DNA replication?

A

DnaB

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11
Q

conditional lethal mutants

A
  • temperature sensitive mutants
  • replication at permissive conditions but fail to function at nonpermissive conditions - like at very high temps
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12
Q

quick stop mutants

A

immediate stop in replication
- elongation polymerase defective and defects precursors
- dna genes

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13
Q

slow-stop mutants

A

-defective in initiation at the origin
- smaller class of dna genes
- DnaA and SeqA

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14
Q

in-vitro complementation systems

A
  • combine extracts from mutants and wild-type strains
  • can add back purified proteins to identify function of a specific dna gene product
  • heat extract from a conditional mutant and assay in vitro
  • add back purified fractions from a wild-type extract
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15
Q

how is DNA synthesized in both replication and repair reactions?

A
  • both use semiconservative
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16
Q

how many bacterial DNA polymerases undertakes semiconservative replication?

A

one, the others are involved in repair reactions

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17
Q

DNA polymerase 1

A
  • encoded by polA
  • major repair enzyme for damaged DNA
  • plays a secondary role in semiconservative replication
  • 400/cell - most abundant
  • full length DNA pol 1 is used for in vitro labeling of DNA by nick translation
  • molecular mass of 103 kD
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18
Q

DNA polymerase 2

A
  • encoded by polB
  • replication restart
  • minor DNA repair enzyme
  • molecular mass of 90 kDa
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19
Q

DNA polymerase 3

A
  • encoded by polC
  • replicase
  • 20/cell
  • contains many subunits
  • molecular mass of 900 kDa
  • has no 5’ to 3’ exonuclease activity
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20
Q

DNA polymerase 4

A
  • encoded by dinB
  • translesion replication
  • SOS repair enzyme of damaged DNA
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21
Q

DNA polymerase 5

A
  • encoded by umuC’2C
  • translesion replication
  • SOS repair enzyme of damaged DNA
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22
Q

how many replicases do eukaryotic nucleus’ have?

A

2

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23
Q

how many replicases do mitochondria and chloroplasts have?

A

a single unique DNA polymerase is required for replication
- other DNA polymerases involved in ancillary or repair activities

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24
Q

what is the common structure many DNA polymerases have?

A

right-hand model
- thumb, palm, finger, N-terminal domain, and exonuclease domain
- the thumb and finger differ between the families
- DNA binds in the cleft between thumb and fingers

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25
Q

fingers

A
  • position template at the active site
  • O helices form the fingers
  • the O helix rotates 60 degrees towards the palm to recruit the incoming dNTP and form one wall of the catalytic site
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26
Q

thumb

A
  • binds DNA as it exists
  • processivity
  • rotates towards the palm by 8 degrees - these changes are cyclonical
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27
Q

palm

A
  • most conserved
  • provides catalytic active site
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28
Q

how does the enzyme recognize a mismatch incorporation?

A

the finger regions in contact with the mismatched base are unable to open to receive the next nucleotide. the DNA then rotates and delivers the mismatch to the 3’ - 5’ exonuclease site

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29
Q

nick translation

A

initiates at nicks in DNA
- extends the 3’ OH end while removing the strand in front by its 5’ to 3’ exonuclease activity
- displaces the existing strand

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30
Q

what are the experimental uses of the klenow fragment?

A

fill-in reaction
DNA sequencing

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31
Q

fill-in reaction

A

used to label recessed ends of DNA
- it can also be used to create a blunt-end for cloning
- this reaction is used to create blunt ends on fragments created by cleavage with restriction enzymes that leave 5’ overhangs

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32
Q

digesting away protruding 3’ overhangs

A
  • another method for producing blunt ends on DNA
  • each generated from restriction enzymes that cleave to produce 3’ overhangs
  • the 3’ - 5’ exonuclease activity of klenow will digest away the protruding 3’ overhang
  • removal of nucleotides from the 3’ ends will continue, but in the presence of nucleotides, the polymerase activity will balance the exonuclease activity, yielding blunt ends
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33
Q

what are the in-vivo functions of DNA pol 1?

A
  • filling in short stretches of single-stranded DNA that arise from DNA replication of the lagging strand or DNA repair when damaged bases have been removed
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34
Q

what is the factor the fidelity of replication is improved by?

A

proofreading by a factor of 100 (100-500) but this is an average

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35
Q

what is the fidelity of DNA synthesis?

A

10^-8 to 10^-10

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36
Q

what is the error rate of DNA synthesis?

A

1 error per genome (4,200 kb) per 100 bacterial replications
about 10^-6 / gene per generation

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37
Q

what proofreading function do all bacterial polymerases have?

A

all bacterial DNA polymerases have 3’ to 5’ exonuclease activity
- operates in the reverse direction from synthesis

38
Q

alpha primase

A
  • priming of both strands
  • no 3’ - 5’ exo
39
Q

delta replicase

A
  • elongation of lagging strands
  • yes, has 3’ - 5’ exo
  • needs the auxillary proteins = RF-C and PCNA
  • high fidelity
40
Q

epsilon replicase

A
  • elongation of leading strand
  • yes, has 3’ - 5; exo
  • needs the auxillary proteins = RF-C and PCNA
  • high fidelity
41
Q

beta

A
  • repair
  • no 3’ - 5’ exo
42
Q

gamma

A
  • mitochondrial replication
  • yes, has 3’ - 5’ exo
43
Q

which enzyme primes lagging strand synthesis in eukaryotes?

44
Q

RF-C

A

replication factor C
- known as clamp loader
- loads PCNA (clamp onto DNA)

45
Q

PCNA

A
  • binds to epsilon and delta to makes them stable on DNA
  • trimer of identical subunits
46
Q

okazaki fragments

A

fragments derived from lagging strand synthesis; usually 1000-2000 bp in length.

47
Q

pseudo-okazaki fragments

A

fragments derived from the leading strand due to misincorporation of uracil. Gapped and waiting for repair.

48
Q

what does DnaB not do?

A

cannot open dsDNA, can only unwinds, DNA that has been already been opened (by DnaA and ATP)

49
Q

what does replication require?

A

requires a helicase to separate the strands of DNA using energy provided by hydrolysis of ATP

50
Q

what is required to maintain separated strands in PhiX models?

51
Q

what can the priming end be provided by?

A
  • an RNA primer
  • a nick in DNA
  • a priming protein
52
Q

where are the two types of priming reaction locations in E. coli?

A
  • the bacterial origin (oriC)
  • PhiX origin
53
Q

what are the implication of cooperative binding in SSBP?

A

most SSB will bind to ssDNA that already has SSB bounds, like at active replication forks

54
Q

pas

A

primosome assembly site

55
Q

recognition complex

A

PriA/B/C/dnaT

56
Q

what are the components of the PhiX primosome?

A

-PriA*
- PriB
- PriC
- DnaT
- DnaB*
- DnaC
- DnaG*

57
Q

what are the roles of PriA?

A
  • translocates along the ssDNA to locate site (pas) by dislocating SSBP
  • loads DnaB onto the origin
58
Q

how does DnaG associate with the primosome?

A

transiently

59
Q

primosome

A

initiates synthesis of a DNA strand
- assemble at the replication fork and do not exist as free complexes

60
Q

replisome

A

complex of proteins engaged in elongation of the newly synthesized DNA strand
- assemble at the replication fork and do not exist as free complexes

61
Q

what are the components used in the PhiX primosome normally used for in E. coli?

A

used to reinitiate at lesions

62
Q

which protein is used to prime PhiX and E. coli lagging strand?

63
Q

what is the monomeric sununits of DNA polymerase 3?

A
  • a catalytic core (alpha, epsilon, and theta)
  • dimerization subunit (tau)
  • a processivity component ( beta clamp)
64
Q

DNA pol 3 -epsilon

A

proofreading

65
Q

DNA pol 3 - theta

A

structural - holds together

66
Q

DNA pol 3 - alpha

67
Q

in E. coli, how does the replisome handle lesions in the template that block DNA polymerase?

A
  • a replication fork stalls when it arrives at damaged DNA
  • after the damage has been repaired, the primosome is required to reinitiate replication
  • the replication fork stalls and may collapse when it reaches a damaged base or a nick in DNA
68
Q

what happens under normal conditions when repairs are made?

A
  • replisomes bypass template damage
  • under normal conditions, DNA pol2 repairs the damaged section and DNA pol 3 continues. Dna pol 2 induced within 2 mins of damage
69
Q

how long does it take to induce pol 4 and 5?

A

induced 50 minutes after damage

70
Q

compare eukaryotes and prokaryotes: helicase

A
  • MCM complex
  • DnaB
71
Q

compare eukaryotes and prokaryotes:: loading helicase/primase

72
Q

compare eukaryotes and prokaryotes: single strand maintentance

73
Q

compare eukaryotes and prokaryotes: priming

A
  • alpha
  • DnaG
74
Q

compare eukaryotes and prokaryotes: sliding clamp

75
Q

compare eukaryotes and prokaryotes: clamp loading

A
  • RFC
  • gamma-delta complex
76
Q

compare eukaryotes and prokaryotes: catalysis

A
  • delta and epsilon
  • pol 3 core
77
Q

compare eukaryotes and prokaryotes: holoenzyme dimerization

A
  • unknown
  • tau
78
Q

compare eukaryotes and prokaryotes: RNA removal

A
  • FEN1
  • Pol 1
79
Q

compare eukaryotes and prokaryotes: ligation

A
  • ligase 1
  • ligase
80
Q

in eukaryotes, there is no DNA polymerase that has 5’ to 3’ nick translation activity, how is the RNA primer in the lagging strand removed?

A

the FEN1 nuclease cuts the primer overhang created by synthesis of the next okazaki fragment

81
Q

FEN1

A

an exo/endonuclease that recognizes the structure created when on strand of DNA is displaced from a duplex as a flap

82
Q

what is the process of general bacteria replication?

A
  • DnaA must be bound to ATP in order to bind to the origin
  • the origin is composed of three 13mers and four 9mers
  • DnaA binds to 9mers first them to 13mers and is able to separate the strands
  • topoisomerase or gyrase relaxes the strands
  • now DnaB/C can bind once there is melting from 30-40 copies of DnaA
  • DnaB is a hexamer that surrounds ss DNA and moves in one direction that propels the replication fork
  • for DnaB to surround. 6 subunits of DnaC is needed to guide and act as a chaperone
  • gyrase, and hu proteins are also needed
  • SSBP bind to prevent ssDNA degradation in the cytoplasm
  • DnaG is the primase that allows pol 3 to bind
  • clamp loader places beta clamp on DNA and ATP is burned
  • the clamp forms around the ds nucleic acid
  • the clamp associated with the core on the lagging strand dissociates at the end of each okazaki fragment and reassembles for the next fragment
  • the helicase DnAB is responsible for interacting with the primase DnaG to initiate each okazaki fragment
  • DnaG is displaced by incoming clamp
  • clamp loader is displaced by DNA pol 3 and beta clamp binds to dsDNA and pol 3
83
Q

Which DNA polymerases can read through thymidine dimers?

A

DNA polymerase 2

84
Q

Know the process of re-initiation DNA synthesis at lesions.

A

Lesions are a severe mutation like a thymine dimer
Have to re-prime
Pol 3 comes off, then pol 4 and pol 5 bind
Then pol 4 and 5 are replaced by pol 3
To re-prime the primosome is needed to recruit DnaB and pol 3

85
Q

Which proteins re-initiate synthesis at lesions and which DNA polymerases can read through errors in replication?

A

Pol 4 and 5 can read through errors
Primosome has to re-initiate replication

86
Q

What kind of primer does alpha use to initiate DNA replication?

87
Q

iDNA

A

short RNA-DNA primer

88
Q

which Phi X model is used to demonstrate lagging strand synthesis? leading strand synthesis?

A
  • replicative form
  • rolling circle
89
Q

Phi X model for leading strand synthesis

A
  • cut both the negative and positive strands - both are not usually nicked
  • this generates two linear ends
  • a helicase is needed to separate strands using ATP to provide energy
  • SSBP is keep DNA in tact
90
Q

PhiX model for leading strand synthesis - rolling circle

A
  • protein A nicks at the origin (pas)
  • links 5’ end of positive strand
  • SSBP keeps DNA ss, binding is highly cooperative
  • Rep provides helicase function and is provided by the host
  • DNA pol 3 elongates the 3’ end of the nick
91
Q

PhiX model for lagging strand synthesis

A
  • primosome assembles at pas
  • PriA along with other proteins bind to pas
  • SSBP is displaced PriA
  • DnaB is recruited by PriA
  • pas hairpin forms
  • primase recruited by DnaB
  • short RNA primers synthesized in opposite direction from primosome movement
  • primers extended by DNA pol 3
  • RNA primers removed by DNA pol 1 and fragments joined by ligase

Molecular Genetics (28 decks)

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