Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOL332 > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

Mendelson + Stahl (Overall)

A

“most beautiful experiment in biology” – shows semi-conservative replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How does DNA replicate (overall)

A

Replicates in semi-conservative fashion

dsDNA seperates –> new DNA is synthesized onto each template

After synthesis – each dsDNA has one strand of newly sythesized DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Imortance of DNA replication

A

Essential for life – basic mechanisms are shared in all living organisms

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Bacteria vs. Euk replication

A

Main differnece = bacteria is circular genomes = need mechanism to make closed circle of DNA Vs. Euk have linear chromsomes (have different mechanims to replicate linear telemere sequance at the end of chromsomes)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Replication phases

A

3 Phases

  1. Initiation –> Unwind DNA + Find starting point + build replication form
  2. Elongation –> DNA is replicated
  3. Termination –> Finish replication
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Bacteria Start of replication

A

Bacteria – DNA is compressed through supercoiling –> topisomerases enzyme that over/under wind DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Intiation

A

Gyrase

Decide where to start –> once unwind = can access DNA –> ORI SITE

DNA A –> Get ssDNA

Biuld replication form – Helicase
- DNA B (Hleicase) = loaded to ss of DNA with help of DNA C (DNA helicase loader)
- Helicased = loaded onto each ddDNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Gyrase

A

A specific Topisomerase that unwinds supercoild DNA for DNA replications
- Removes + supercoiling

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How does Gyrase bind to DNA

A

To unwind – Gyrase = binds to sequens of DNA –> cuts through dsDNA and unwinds one loop = shifts DNA molecule from one domain of the enzyme to another domain of enzyme –> then gyrase glues ends back

Requires ATP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What does Gyrase require

A

Requires energy in the form of ATP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What inhibits Gyrase

A

Classes of Antibiotics “quinoloes” = prevents bacteria DNA replication by targeting ATP binding sites of Gyrase

Ex. Naladixic acid

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Origin of replication

A

Region of DNA that is origin of replication = ori site

Bacteria = only one ori site on circular chromosome

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Ori site

A

Origin of replication

Conatins 3 replication regions that have high number of A/Ts –> AT rich region = followed by 9 BP seq

9 BP seq = DNA A box

Intersperesed through 275 ori sequence = repeats of 4 BP GATC

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

DNA A box

A

9 BP sequence after Ori site

repeats 5 times in E.coli

DNA A recogniztion sequence

GATC sequence – C must be methylated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

GATC in Ori

A

Interspersed in ori sequence = repeats of GATC

For DAN replication to occur C in GATC = chemocally odifies through methy group

C = methylated on both strands –> methyl groups help porteins reconnize ori sequnce

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

DNA A

A

DNA binding protein that scans genome looking for DNA A box –> When DNA A binds to DNA box = causes dsDNA to bend
- DNA A causes tension to DNA – H bonds are broken in AT rich region of Ori sequence = seperate dsDNA

When many DNA A proteins are bound to ori site = get bending of helix structure = creates physical stress –. To releive stress – H bonds between A-T base pairs and AT sequnces in DNA A box are broken = dsDNA seperates = get ssDNA to act as tenmplate for replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Helicase

A

DNA B – Multimerix complex shapes like donut

To get around ssDNA DNA C binds to DNA A and to Helicase –> Seperates Helicase ring –> wraps the helicase around DNA and then reforms donut ring

DNA B = seperates dsDNA (unzips 5’ - 3’ to get more ssDNA for replication
- Unwinds replication form
- Creates tension in dsDNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

DNA C

A

DNA helicase loader

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Movement of Helicase

A

Moves down ssDNA 5’ –> 3’ so each helicase moves in opposite directions

Movement of Helicase = dislodges DNA and DNA C from ori sequnece because DNA C is not needed once the Helicase is loaded

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Replication Bubble

A

Structure created in intiation

Conatins 2 replication forks

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Replication forks

A

Site where ssDNA is being seperated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Stability of ssDNA + ssDNA binding proteins

A

ssDNA is NOT stable – will try to form dsDNA –> To prevent dsDNA from reforming ssDNA binding proteins bind non-specifically to ssDNA and prevent H-bonds from reforming between 2 ssDNA
- Allows bubble to get bigger + DNA helicase to do its jobs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Gyrase + Helicase

A

DNA Gyrase = unqinds the supercoiled DNA caused by helicase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Electron micropscope images of DNA replication

A

Shows – chrmosomes activley replicating

Shows:
1. Two replication forks in bubbles
2. In bubble have new DNA synthesized from template DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Elongation

A

DNA replicates during Elongation

Overall – Primer binds + replication assmebled

DNA polymerase

Primase –> Get RNA primer

Onece primer is formed = DNA polymerase is recurted

DNA polymerase creates new strands of DNA

Reprisome holds DNA polymerase to allow new DNA to be synthesized

End = close gaps in okazaki fragments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Enzyme responsible for DNA replication

A

DNA polymerase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

DNA polymerase

A

Enzyme responsible for DNA replication

Binds to ssDNA –> Wraps around DNA

Main function = add free nucleotides to 3’OH end of DNA chain (only add to 3’ end = DNA replication occurs 5’ - 3’)
- Continue adding completely nucleotides (complementary to template)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

DNA pol binding to DNA

A

Binds loossley because needs to move down DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Limitation of DNA polymerase

A

Only adds nulceotides to existing chain – can’t begin DNA replication from scratch

Because can’t begin without free 3’ end of nucleotide chains to build = the first steo in elongation requires sequnece of startong oligionucelotiodes = “primer”

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

DNA pol in E.coli

A

5 types of DNA polymerase – main type is DNA polymerase 3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Start of elongation

A

Because can’t begin without free 3’ end of nucleotide chains to build = the first steo in elongation requires sequnece of startong oligionucelotiodes = “primer”

Primase = Synthesizes RNA primer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

Primase

A

Synthesizes RNA primer –> Can intiate RNA synthesis without existing oligionucelotide strand

Binds to DNA helicase and scnas replication bubble until finds specific target sequnce on ssDNA

When primase finds target –> Uses it as a template for RNA primer synthesis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Primasome

A

Helicase + Primase complex

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

How does DNA polymerase hold onto template

A

DNA polymerase holds onto template DNA by sliding clamp + Sliding clamp loader

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

Sliding Clamp + Sliding clamp loader

A

Holds DNA pol. onto template
- DNA polymerase is loaded onto ssDNA by sliding clamp and sliding clamp loader

tethers 2 DNA polymerase on each strand –> Ensures replication occurs on each strand simultaneously

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

Replisome

A

Complex of Helicase + Primase + DNA pol + Sliding clamp + Loader

DNA replication occurs at replisome

Creating replisome – DNA polymerase is loaded onto ssDNA by sliding clamp + Sliding clamp loader

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

DNA polymerase (function)

A

Creates new strands of DNA

Adds free nucleotides to 3’ ends of RNA primer in 5’ –> 3’
- Holds onto template and adds nucleotodes to new strands

helps reshape DNA helix by aiding in H-bond formation between bases in template strands and new DNA strand

Can also proofread + correct mistakes that is made

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

Rate of DNA polymerase

A

750 BP/second – very fast

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

DNA polymerase mistakes

A

Sometimes polymerase makes mistakes –0 adds incoreect nucelotides onto new strand

1 Mistake/10,000 BP

Polymerase = can sometimes fix mistakes –> Halt polymerase activity THEn relax the dsDNA being fromed = expose new templates + new domain of enzyme –> Exonucleose activity = chews up newly synthesized DNA and DNA polymerase restartes adding new nucleotides back on chain (exonuceloase is 3’ - 5’)

After DNA fixes mistakes – 1 mistake in 10^-4 BP

DNA polymerase = very accurate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

Fidelity

A

Acurracey

DNA polymerase = very accurate = high fidelity – have very few mistakes in newly sythesized DNA strands

41
Q

Leading vs. Lagging strand

A

Leading Strand = On one template strand DNA replication is in smae direction as the helicase
- DNA synthesis occurs continously

Lagging Strand = DNA synthesis is in opposite direction from reprisome
- DNA replication offcurs until polymerase bumps into RNA primer
- DNA synthesis = discontinous = get okazaki fragments
- Discontinous

42
Q

Lagging strand end

A

After replication – Lagging strand has bits of DNA/RNA + breaks in DNA phsphate backbine where 5’-3’ ends of phosphate are not covalently bonded

43
Q

Closing gaps on Okazaki fragments

A

END of elongation

Difefrent DNA polymerase (DNA pol. 1) –> recognizes DNA/RNA hybrids – attatches there and cleaves RNA compoennt using exonuclease activity

Polymerase ALSO fills in gaps using DNA nucleotides

Ligase = connects phosphate backbones

44
Q

Ligase

A

COnnects phosphate backbone = get continous DNA strand

45
Q

How longs does Elongation go

A

Elongation occurs until all DNA on chromsome has been replicated

46
Q

Where does DNA replication occur

A

Occurs at each replication fork –> bubble gets larger and larger until entire circulare chromosome has been replicated

47
Q

Timing in replication

A

It is impirtant that replication forks end at the same time or else DNA replication will just keep going in circle

Replisome will keep going in circle forever

Issue = replication forks might not finish at the same time = need mechanisms to ensure that replication stops as get haf way around the circle

48
Q

How to stop replication

A

Done through Tus/ter sequence on oppositre sides of Ori sequnece

49
Q

Tus/Ter sequnece

A

Termination sequnece

Has 2 domains – ter 1 and Ter 2

Tus = binds to ter sequnce
- Tus binds to Ter 1 –> Tus binds to one Ter on top of DNA strand and Another Tus binds to Ter 2 on bottom strands

Tus/Ter complexes = dorectional

50
Q

Direction of Tus/Ter

A

Tus/Ter complexes = directional

ONe lines to top of DNA strand and other tp bottom of DNA strand –> Directionality helps guide termination

Example
Ter binds to top of DNA strand –> Replication fork approaches Tus/Ter from either direction (from the top or the bottom)

When Helicase appraches Tus/Ter ocmplex on the same strand that the Tus is bound to (Tus is on top and Helicase comes on the stop strand) = NON-Permissive position –> Helicase is blocksed = whole replisome sits and waits

IF helicase appraches from opposite side (Helicase on top and Tus on bottoms) –> helicase can remove Tus from the complex and continue replicating

At one Tus/ter the helicase is blocked and other noth blocked –> eventually the two replication forms will meet at one Tus/ter complex
- Which depends on which fork gets there first

51
Q

Euk replication

A

Similar to Prokaryotic – same ideas just different names

52
Q

Intiation (Euk)

A

Euk chromsomes = longer = in order to replicate in suffeincet time = have intiation in multiple positions across linear chromsome

53
Q

Orgin of replication (Euk)

A

ARS – Autidory replication sequnece

Euk chromosomes = have multiple ARS

Each ARS = can intiate replication bubble to begin synthesis

In ARS –> recocgnized by ORC (orgin of replication) –> recruits other enzymes to make PreRC (pre replication complex
- Provides location/permission of DNA replication to occur

54
Q

Goal of initiation in Euk

A

Goal of intiation in Euk = same as prokaryotes –> open dsDNA to provide template for DNA replication

55
Q

Elongation (Euk)

A

Euk = more complicated than bacteria

Have more than 10 DNA polymerase –> Difefrent polymerase for leading/lagging strand

56
Q

Unwinding DNA in Euk

A

Unwind dsDNA – more complex because DNA is round around histones to produce nucleosomes

Nucloesomes need to be diseembled and immediatley resembled following replication (Disembled + Reasembles siumlatneous)

57
Q

Key differences between Prok and Euk

A

Termination

58
Q

Termination of linear ends of chromosomes

A

Telemerse sequence – end of telemerse = has region of ssDNA on 3’

Have 3’overhange – probelm for DNA replication

DNA = 5’ –> 3;’ – only way to replicate DNA in overhang is if habe primer sequnce to the right of 3’ end
- No primer for DNA polymerase = overhand won’t be replicated = with each round of replication chrosmome would get shorter

59
Q

How do Euk cell replicate the telemere

A

Telmerase

60
Q

Telemerase

A

Ribozyme (enzyme that has protein Subunit and RNA components)
- RNA component = complemnets to overhang = allopw telermase to bind to end of telemere

After binding RNA compoennets still conatins ss domeain

Telemerase = used own RNA compoenent as template for DNA synthesis adding nucelotides to 3’ overahnd = extends ends of chrosmomes

***Telermerase = sepcial kind of DNA polymerase

Binds to overhand and synthesized DNA complememtary to RNA –: extends the length of hthe chromsomes to ensure the overhang can be replicated

Makes telemer sequence longer

61
Q

If telermases exteneds length of chromsome why fo telemerse get shorter during aging process

A

Germ line cells express lots of telermase so chrosmomes ends up in gametes have long telemers

Somatic cells = express less telermase
- Without telermase chrosmomes do get shirter each round of replication
- Age = increase rounds of replication = shoter telemers

62
Q

Increasing teloermase as age defying porcess

A

Might say that increasing telermase in cells could be age defying process

Issue = some cancer cells avoid cell death by expressing high levels of telermase = telermase might not act as perfect fountain of youth

63
Q

Use of Telemerase

A

Allows chromosomes length to be miantained from one generation to the next

Maintain length in species

64
Q

Structure of DNA + DNA replication

A

The structure of DNA provided a way to understand how DNA can be copied during inheritance

65
Q

Structure of DNA

A

Helix

A-T

G-C

BP facing in

66
Q

Copying mechanism of DNA

A

1 peice of DNA –> 2 peices of DNA

DNA replication = semi-conservative replication

67
Q

Methods proposed for DNA replicatons

A
  1. Conservative – Old –> get old + new
  2. Semi-conservative
    • Know this is true
    • DNA –> Pulled apart –> DNA synthesis on each strand
  3. Dispersive
    • Mixture of chunkcs of old + new scattered
    • No one thought this is what actually happens but posibility
68
Q

Meselson + Stahl expeirmnent

A

Idea – First grow bacteria in N15 so that all DNA is heavy –> THEN shift to N14 (light)
- N15 = radiolabeled (heavy)

Looking at what happens to band

Start = All DNA made have N15 –> THEN run on gradietnt gel

69
Q
A

1 – Dispersive (would just be a mess)

2 – Semi conservative

3 – Conservative – band for heavy + band for light

70
Q

Expected results from Stahld Experiment

A

After two rounds:
If conservative – Always have heavy –> Get more light but no middle band

If semi-conservative –> Still distict bands L/H but start to get more light

Dispersive –> Still smear

71
Q

Gel in Stahl

A

After 40 Minutes – ALL middle (no heavy)

As replicate more – newly synthezied used N14 = get light eventually all light + some middle
- Middle never goes away (Always there)

72
Q

Goal + end product of each Phase

A

Intiation – Unwind DNA – DNA = supercoiled –> need to get ssDNA to copy it
- require energy
- get ssDNA template

End product = ssDNA = can replicate
Goal = create ssDNA
End = replication forks

Elongation
Goal = synthesize new DNA
End = have newly syntheszed DNA (Nascant DNA)
- Bacteria –> Linear fragment
Goal: Repliacte Template DNA
End: Linear dsDNA

Termination
- Goal = finish replication
- End = get dsDNA end prodyct

Goal : Stop replicating DNA + glue to get circle
End = get circular DNA

73
Q

List things needed for each step (in order) + State general use of each

A

Initiation:
1. Gryase –> Unwinds supercoil
2. Energy (ATP) –> Energy to unwind
3. Ori Sew –> Where DNA A binds – Replication starts at Ori Seq
4. DNA A –> binds to DNA A box
5. DNA A box –> Part of Ori Seq
6. DNA B –> Helicase – keep unwindng DNA
7. ss Binding proteins –> Stops dsDNA from reforming
8. DNA C –> helicase loader
9. Template DNA

Elongation:
1. primer
2. DNA pol.
3. Sliding Clamp
4. Template DNA
5. Primers
6. Primase –> creates primer
- pOlymerase can’t start from nothing – need free end to add nuceloties to
- Primase = builds RAN chain from nothing
7. Sliding clamps loader
8. Free nucleotodes
9. Energy (ATP)
10. Ligase

Termination:
1. Tus
2. Ter
3. Energy
4. Ligase

74
Q

Intiatian

A

DNA box bind to ori = caises to bend = tension = pops open = get ssDNA

To load helicase on = cut open –> wrao around –> hook toegtehr again (loaded by DNA B loader (DNA C)

75
Q

Elongation

A

Have fork –> recurit primase + recurit Pol –> Build DNA chain

Helicase recruits primase THEN DNA polymerase = get nascant DNA
- Held together with slding clamp

76
Q

H bonds between BP

A

A-T –> 2 H bonds –> region in ori is AT rich – where dsDNA opens to be ssDNA

G-C –> 3 H bonds

77
Q

Direction of replication

A

5’ –> 3’

Leader strand – Towards fork

Lagging strand – Away from fork
- Goes until bumps into primer –> get okazaki fragments

78
Q

Fidelity of DNA Pol.

A

DNA pol. = high fodelity

Might make mistakes but can correct BUT somtimes mistakes get through

79
Q

Termination (bacteria)

A

Replication forms will meet –> won’t meet at exctley the dame time

If didn’t meet at the same point = would keep going in cirvle = need to end at the same time –> does so using the Tus/Ter

Tus binds to Ter –> have 2 Ter sequenceies (one on top and one on bottom)
- If Tus is on the top strand and helicase is on top = helicase is blocked BUT if helicase comes from the bottom and Tus is on top = hits the Tus off – will be blocked by other tus

END – one will be blocked until other catches up
- End = ligate 2 fragments together

80
Q
A

If kept at high heat = wouldn’t need ss Binding proteins

81
Q
A

Don’t need ligase if making short thing

82
Q
A

Don’t need any of them

83
Q

What do we need to make short DNA

A

DNA
Primer
Nucelotides
Energy

84
Q

DNA replication in test tube (not PCR)

A

1 single stranded template creates 1 dsDNA product

Heat = get ssDNA

Add primer + DNA polymerase = get new DNA

END = get 1 ssDNA and 1 dsDNA

DNA polymerase falls apart when the temeprature is raised – need a new polymerase to make a second round of synthesis

85
Q

What do you need for 2 rounds of DNA synthesis in a test tube

A

DNA polymerase falls apart when the temeprature is raised – need a new polymerase to make a second round of synthesis

86
Q

Sanger sequncing

A

Sanger = invented method of sequnecing DNA

Small qunatities of radiolabeled nucleotides that blcoked DNA sythesis were added to synthesis reaction
- Add nuloetides that stop chain seq.

The synthesis mictire would contain difefrent lengths of DNA depending where the ddNTP was incorportaes – these different lengths would be seperated by a gel

By Running difefrent reactions with different ssDTPs the sequence could be “read” dorectlet from the gel

***Need 4 tubes

87
Q

Issue in sanger seq

A

Slow + can only sequencey short regions at a time
- Can’t multiplex reactions
- Would need 1 million reactions to sequnce DNA)

88
Q

Results of Sanger

A

See gel

Ex. – have end # of Gs OR get band that ends in C

89
Q

Chain termination by ddNTPs

A

If have ddNTP = no OH = stop extension
- Add ddNTP = can’t get next nucleootide

Fluorernstley label ddNTPs

90
Q

Change in sequencing

A

1977 – radiolabeled ddNTPs + Gels

2023 – Flourecent ddNTPs + capilary tubes/compures

Sanger = the most accuarte methods of DNA seq

91
Q

Importance of PCR

A

Considered one of the most important scientific advances in molecular biology

92
Q

Kary Mullis

A

Invented PCR –> Was a biochemist working on chemical synthesis of oligios (primers)

Likes surfing + Skiiing + LSD + WOmen

After his BS = he spent a couple of years writting fiction

During grad school he took leave and managed a bakery

Synthesized recreational drugs in spare time

FOUND – that if he used 2 prismers and a DNA polymerase that could wistand high temperatires he could amplify the amount of DNA sunthesized exposnetially

93
Q

What did they need to make PCR

A

At time = they got bacteria from hot vent –> proteins don’t fall apart at high temperatures

Kullis = realized that he could have 2 primers + use DNA polymerase taht doesn’t fall apart when hot = could have more than one round of replication

94
Q

PCR replication

A

dsDNA –> Heat –> prmer binds –> New DNA is synthesized on top of old DNA –> keep doing cycle

Exponential growth of DNA

95
Q

Kary mullis was…

A

Intolerable genius – fought with nearly everyone (bosses +_ labmates _ security guards + receptionists)

He turned his back on scinece

Arugues that AIDS and climate chnage were hoaxes

96
Q

Importance of PCR

A

Medicine + Forensices + Agricultiure + Infectious disease + research

97
Q

Steps of PCR

A

LOOK AT Pre-class (REAL)

98
Q
A

Answer: B (Need inwards)

Primer = 5’ –> 3’

BIOL332 (25 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions