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MCBM EXAM 5 DANIELLE > DNA REPLICATION > Flashcards

DNA REPLICATION Flashcards

1
Q

What is semiconservative replication of DNA?

A

the 2 original strands of DNA separate and each acts as a template on which a new complementary strand is laid down
(storage and transmission of genetic information)

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2
Q

Who proved DNA replication is semiconservative?

A

Meselson and Stahl (1957)

used ecoli and heavy nitrogen

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3
Q

What did Kornberg do in 1958?

A

descried the purification of an enzyme called DNA polymerase I from Ecoli that synthesized DNA

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4
Q

How is DNA synthesized?

A

from nucleotide triphosphate precursors by the enzyme DNA polymerase

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5
Q

What did John Cairns in 1963 reveal?

A

that an ecoli chromosome is a single 1.7mm circular molecule (replicating DNA looks like theta)

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6
Q

How is DNA replicated in bacteria? (remember circular structure)

A

bidirectionally because a replication fork is found on each end of the loop where DNA is unwound and the daughter strand is being synthesized
(don’t worry too much about this)

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7
Q

What is a primer?

A

a segment of nucleic acid (RNA or DNA) base paired to the template and with a free 3’-OH

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8
Q

Who discovered the DNA had a template strand and base pairing rules?

A

Watson-Crick

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9
Q

What is processivity?

A

number of bases synthesized before the DNA polymerase enzyme dissociates from the template (can be thousands)

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10
Q

What does DNA polymerase exhibit?

A

processivity

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11
Q

Is DNA polymerase and DNA replication accurate?

A

yes very accurate

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12
Q

Is DNA replication more accurate in humans or bacteria?

A

bacteria

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13
Q

What improves accuracy of DNA polymerase and DNA replication?

A

mismatch repair

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14
Q

What causes tautomers to form?

A

chemical structure is not absolutely stable, proton rearrangement leads to different chemical forms

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15
Q

What are tautomers?

A

the same atomic composition with a slightly different chemical structure (changes the pairing characteristics of specific bases)

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16
Q

At any given time roughly how many tautomers are there?

A

1 in 10,000 molecules are in rare tautomeric form

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17
Q

A cytosine tautomer base pairs accurately with what?

A

adenine (instead of guanine like it would if it was not a tautomer)

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18
Q

What is the proofreading activity of DNA polymerase I?

A

3’ to 5’ exonuclease activity

acts ONLY on the base just incorporated (no others)

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19
Q

Does polymerization activity stop when there is an error found?

A

yes because it has to stop and fix the problem (remove mispaired nucleotide) then it will resume

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20
Q

How many E coli DNA polymerases are there and what are they?

A

3
DNA polymerase I
DNA polymerase II
DNA polymerase III

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21
Q

What is the function of DNA polymerase I in Ecoli?

A

mostly for repair

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22
Q

What is the function of DNA polymerase II in Ecoli?

A

(specialized repair function) don’t need to know

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23
Q

What is the function of DNA polymerase III in Ecoli?

A

mismatch repair (main DNA replication enzyme)

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24
Q

Which DNA polymerase in Ecoli is very processive?

A

DNA polymerase III

once it clamps on the DNA it stays on for a long time and adds over half a million bases before it is removed

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25
Q

How many subunits does DNA polymerase III have?

A

10

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26
Q

What are the 3 genes that encode the 3 DNA polymerases?

A

polA (correlates with DNA polymerase I), polB (correlates with DNA polymerase II), polC(correlates with DNA polymerase III)

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27
Q

What is the activity of polA and what exactly does it do?

A

has a 5’ to 3’ exonuclease activity used to get rid of primer RNA during DNA replication

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28
Q

Is polA, polB, or polC the large gene that makes a big donut shaped clamp?

A

polC

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29
Q

What are the 3 stages of DNA replication?

A

initiation
elongation
termination

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30
Q

Which stage in DNA replication most complicated?

A

initiation

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31
Q

Where does DNA replication begin?

A

origin of replication (ORI)

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32
Q

How many proteins are involved in DNA replication in Ecoli and is it more or less than in humans?

A

about 20 which is more than what is used for humans

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33
Q

What is DUE? (in Ecoli)

A

a 13 base pair sequence that unwinds the DNA to start the process of replication

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34
Q

What does it mean when you see GATC during DNA replication in bacteria?

A

means mismatch repair is taking place at the methylation site which tells us that replication has been initiated at this site (THIS WILL ONLY HAPPEN ONCE)

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35
Q

What are R region (R1-R5) seen in DNA replication in Ecoli?

A

a DnaA protein binding site that has five regions with a 9 base pair sequence

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36
Q

What happens at the R regions (R1-R5)?

A

DnaA protein binds at these 5 sites and causes DUE to open up into single strand portions

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37
Q

What is DnaA?

A

A protein required to initiate replication at the E. Coli origin

38
Q

What are the 8 proteins required to initiate replication at the E. Coli origin?

A 
DnaA
DnaB (helicase)
DnaC
HU
DnaG (primase)
SSB
DNA gyrase
Dam methylase
39
Q

What does DnaB (helicase) do?

A

unwinds DNA to expose a single strand template

40
Q

What does DnaC do?

A

is a helper for DnaB

41
Q

What is HU?

A

histonelike protein (remember bacteria doesn’t have true histones)

42
Q

What does DnaG (primase) do?

A

synthesizes RNA primers

43
Q

What is SSB and what does it do?

A

single strand binding protein- binds single stranded DNA

44
Q

What does DNA gyrase do?

A

relieves torsional strain generated by DNA unwinding

45
Q

What does Dam methylase do?

A

involved in mismatch repair system - will tag the parent strand and tell the fixing mechanism which strand is defective and needs to be fixed

46
Q

Why does the DNA unwind at the DUE sequence?

A

it has a lot of A-T bonds which are not very strong so it is easier to twist open here

47
Q

What 2 things bind to form an initial complex?

A

dnaA and oriC

48
Q

What is the prepriming complex?

A

once DnaA is established DnaB is brought in with DnaC to stabilize the unwound DUE elements

49
Q

What is single stranded DNA stabilized by?

A

single strand binding proteins (SSBs)

50
Q

Do all the steps in the initiation of replication need energy?

A

yes all need ATP

51
Q

What do all synthesizing enzymes require?

A

a primer

52
Q

What is a problem for initiation of DNA replication?

A

primers can only add to the 3’ end of an existing strand and cannot initiate a new strand

53
Q

What 2 things bind to form the replisome?

A

DNA polymerase III and primosome

54
Q

What do replisomes do?

A

move around chromosomes (bidirectional replication from a fixed origin)

55
Q

What is primase?

A

a specialized RNA polymerase that synthesizes short 15 nucleotide stretch of RNA on DNA template which remains base paired

56
Q

What enzyme creates the primer for DNA polymerase?

A

primase

57
Q

Primase binds to DnaB and DnaC on the DNA template (leading strand ) other proteins add on to form a complex called what?

A

primosome

58
Q

What is a problem for DNA elongation?

A

all DNA polymerases add bases to the 3’ end , has NEVER been found to add bases to the 5’end

59
Q

What is the new daughter strand called?

A

leading strand

60
Q

What is associated with the lagging strand?

A

okazaki fragments

61
Q

Why is the lagging strand made in fragments?

A

it is synthesized in short stretches of several hundred to a few thousand bases (okazaki fragments) because of the orientation required by DNA polymerase

62
Q

What happens in front of the replication fork?

A

positive supercoiling

63
Q

What molecules are in charge of relieving the coiling at the replication forks?

A

DNA gyrase and topoisomerase I

they maintain the chromosome negatively supercoiled

64
Q

What happens if DNA gyrase is not working?

A

positive supercoiling stays intact and replication will stop

65
Q

What are DNA gyrase inhibitors?

A

antibiotics that inhibit DNA gyrase and subsequently stop replication

66
Q

How is elongation initiated?

A

by forming a loop of the lagging strand template on the replisome
(loops around so it is in the same direction as the leading strand) - just a small section

67
Q

What begins the synthesis of the lagging strand?

A

second DNA polymerase III molecule that binds to a primer

68
Q

What is found on the 5’ end of each okazaki fragment?

A

an RNA primer

69
Q

Are okazaki fragments covalently linked to each other?

A

no

70
Q

What repairs okazaki fragments and produce a continuous DNA lagging strand?

A

DNA polymerase I and DNA ligase

71
Q

What type of enzyme activity does DNA polymerase I have?

A

5’ to 3’ exonuclease activity

72
Q

How is RNA removed from the 5’ end of the okazaki fragment?

A

by DNA polymerase I and then DNA ligase covalently seals the gaps in the backbone

73
Q

What allows the two double helices to separate? (circular arrangement -still talking about ecoli)

A

toposisomerases

74
Q

How is DNA replication terminated?

A

replication forks meet (not much else is really known)

75
Q

How many polymerases are used in eukaryote DNA replication?

A

at least 5 (1 in mitochondria)

76
Q

Is replication initiated at multiple origins or one origin in eukaryotic DNA replication?

A

multiple origins (roughly every 40-100kb) otherwise chromosome replication would take several days

77
Q

What are some difference in DNA replication of eukaryotes and Ecoli?

A

eukaryotes have linear chromosomes ecoli has a double stranded circle eukaryotes have multiple origins and ecoli only has one

78
Q

What are the 5 DNA polymerases in eukaryotes?

A 
alpha
beta
gamma
delta
epsilon
79
Q

What does alpha do?

A

it is a primase

80
Q

What two eukaryotic polymerases are involved in nuclear replication, nucleotide excision repair (NER) and mismatch repair (MMR)?

A

delta and epsilon

81
Q

What does beta do?

A

mostly used for repair (base excision repair)

82
Q

What does gamma do?

A

involved in mitochondrial replication

83
Q

Why is linear chromosomal DNA a problem for replicating the ends of the DNA molecule in eukaryotes?

A

the end of the lagging strand that has the primer cannot be fixed so it would just cause shorter and shorter chromosomes in each replication cycle

84
Q

What are telomeres and what do they do?

A

telomeres are at the ends of eukaryotic chromosomes and they allow linear molecules to be replicated without loss of information

85
Q

How are telomeres synthesized?

A

without a DNA template by the enzyme telomerase

86
Q

What is telomerase?

A

a specialized reverse transcriptase that has an RNA component to serve as a template for the synthesis of a repetitive GT rich DNA sequence
also attaches chromosomes to the nuclear envelope

87
Q

Can priming occur in the telomere? Can coding of DNA occur here?

A

priming can occur here but coding of DNA cannot

88
Q

What do telomeres prevent?

A

undesirable fusion of chromosomes

avoids aberrant recombination

89
Q

What does it mean for telomeres to serve as a mitotic clock?

A

interesting for cancer research - if telomerase is blocked it can stop replication

90
Q

What is senescence?

A

when cells don’t die they are not replicating but they are just sitting there (shorter telomeres induce this and apoptosis)

91
Q

Is telomerase active in germ cells, stem cells and tumors?

A

yes

MCBM EXAM 5 DANIELLE (6 decks)

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