Chapter 34: DNA Replication Flashcards
1
Q
The process in which the DNA within a cell makes an exact copy of itself.
A
DNA Replication
2
Q
Eukaryotic cells (self-cycle) replicate in what phase?
A
S phase
Must be done rapidly with control and accuracy
3
Q
DNA Replication is considered
A
Highly coordinated
4
Q
The E. coli genome has…
A
4.6 million base pairs
5
Q
The E. coli is replicated in _____, at a rate of _____!
A
less than 40 minutes
Rate of 2000 nucleotides per second
6
Q
Old duplex is conserved and new duplex composed of two completely new strands
A
Conservative
7
Q
Sections of the old duplex dispersed somewhat randomly to the two daughter duplexes
A
Dispersive
8
Q
Single strand of DNA is _____
A
Unstable
9
Q
Base pairs face _____
A
Inward - poses a challenge for replication
10
Q
Conservative replication takes a strand of DNA …
A
opens up the duplex and make a copy and close it back up
Start with original and make an exact copy
11
Q
Dispersive replication is where you end up with
A
a hybrid where you could break the backbone of the DNA and religate it together
“Patchwork”
12
Q
Two daughter duplexes with one each of the old and new strands
A
Semiconservative
13
Q
Semiconservative was proposed by
A
crick and watson start with double stranded, split apart and end up with a copy. Two resulting strands with half of original and after second generation, two new strands
14
Q
N14 is the _____ of nitrogen
A
Natural isotrope
15
Q
Meselson Stahl Experiment
A
Developed a method to Get a very fine density gradient established in the tube and match it to the DNA
Can separate DNA that has N14 from N15 based on density
16
Q
What gets labeled in the Meselson Stahl experiment?
A
Bases get labeled
17
Q
In the Meselson Stahl experiment, First generation results eliminate _____
Second generation eliminates _____
A
First - conservative model
Second - dispersive model
18
Q
New DNA is all grown in the presence of..
A
N14
19
Q
By two generations, you end up with…
A
2 peaks at equal intensity (One peak is 50/50, the other is N14)
20
Q
By the first generation, we know that the _____ model is not correct.
A
Conservative
21
Q
By the second generation, progressively enrich…
A
Towards fully N14 but not with the discreet jump with two different peaks
22
Q
_____ is at the heart of DNA replication
A
DNA Polymerase
Adds nucleotides 1 by 1
Phosphodiester bond formation
23
Q
What are the 4 important characteristics of DNA Synthesis?
A
- Deoxynucleoside triphosphates and Mg2+ are required. dATP, dGTP, dCTP and dTTP
- A template strand is used to direct DNA synthesis.
- Chain extension is always 5’ to 3’
- A primer from which the new strand grows must be present
24
Q
Binding of the _____ induces a structural change, closes over the nucleotide, forming a pocket into which only the correct nucleotide fits.
A
incoming dNTP
25
Specificity is determined by two factors:
1. the correct hydrogen bonding between the incoming dNTP and the DNA template
2. the overall shape of the incoming base
26
Specificity of replication is dictated by the \_\_\_\_\_.
Complementarity of bases
27
DNA Replication requires \_\_\_\_\_.
Highly processive polymerases
28
The holoenzyme responsible for the majority of DNA synthesis in E. coli is \_\_\_\_\_, a multi-protein complex.
DNA polymerase III
29
DNA polymerase III is highly processive - once it begins catalysis it rarely \_\_\_\_.
Releases the DNA substrate.
30
DNA's replication process is...
Semi-conservative
31
In the Meselson-Stahl experiment, how many bands will conservative DNA show?
2 bands
32
Separation in the Meselson-Stahl experiment based on...
Density
33
The Meselson-Stahl experiment was used to determine...
The correct model for replication
34
The reaction catalyzed by DNA Polymerase produces...
1 extra base and the two phosphate groups drive the reaction forward
35
The primer will have a _____ and cleaves...
Hydroxyl group and cleaves the two phosphates of incoming base
36
The induced fit is not efficient, but...
Cannot afford to have errors in DNA, so it is better to have lost a little energy
37
dNTP removes _____ correct BP and will fall into the _____ section.
1/20
Exonuclease section
38
The base must match the \_\_\_\_\_.
Active site
39
1000x more accurate because of...
Polymerase I exonuclease activity
40
A holoenzyme does not require....
Any cofactors to activate
"Whole" how it is
41
DNA Polymerase III almost never...
Stops replicating, process keeps chugging
42
3 distinct stages of DNA replication:
Initiation, elongation and termination
43
Initiation
Adding primer
44
Elongation
Adding bases
45
Termination
Connecting fragments and ridding of primer/proofreading
46
DNA replication is carried out by a large complex containing multiple...
Proteins (\>20)
47
The multi-step process of DNA replication:
Initiation
Helix must be unwound
Primer synthesis
Primer removal
One fork, two antiparallel strands
Connecting the pieces
Proofreading and error correction
48
Replication begins in E. coli at a unique _____ site called the...
245 base pair site called the oriC locus (replication primer)
49
Helicases are _____ and always require...
Unwinders and always require ATP
50
Single-stranded binding protein (SSB) bind to _____ to prevent \_\_\_\_\_.
AT rich sites to prevent reattachment of the helix
51
The initiation protein allows...
Unwinding of the helix
52
RNA polymerase (primase) enables...
DNA synthesis to begin
53
RNA primer adds _____ to serve as primer after DNA is unwound.
10 RNA nucleotides
54
DNA template ----------\>RNA Primer ----------\>Newly synthesized DNA
What fills in the arrows?
1. Primase
2. DNA polymerase
55
What prepares the double helix for unwinding?
Topisomerases
56
When DNA is unwound for replication, _____ occurs due to overwinding in nearby regions of the helix.
Supercoiling
57
Topoisomerases induce OR eliminate...
Supercoils
58
Type 1 topoisomerases...
Eliminate supercoiling
59
Type 2 topoisomerases...
Introduce supercoiling
60
The "negative" supercoiling starts to...
Unwind (Areas between supercoiling are where its unwound
61
Bacterial topoisomerase is a...
Therapeutic target
62
What are two inhibitors of DNA gyrase that are used to treat bacterial infections?
Nalidixic acid and ciprofloxacin
63
What prevents anthrax poisoning?
Ciprofloxacin
64
DNA gyrase is what type of topoisomerase?
Type 2 in bacteria
65
What two things are needed to separate the DNA strands?
Specific helicases and ATP hydrolysis
66
Helicases require _____ and separates _____ to make the DNA available for DNA polymerase.
ATP and separates double helix
67
Helicases have how many subunits?
6
68
Defects in the helicase can result in...
Werner syndrome
69
Werner syndrome causes...
Premature aging
70
The site of replication is called the...
Replication fork
71
The replication fork moves in one direction, so...
Both strands are copied simultaneously
72
All DNA polymerases synthesize DNA only in the...
5' --\> 3' direction
73
Okazaki fragments have...
A primer between each fragment and allow 3' --\> 5'
74
What begins each okazaki fragment?
RNA Primer
75
What is the complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template)?
Primosome
76
Primosome adds your...
Primer
77
DNA polymerase uses the RNA primer to start...
The lagging-strand DNA snythesis
78
A _____ is needed to start each Okazaki fragment.
Primer
79
The lagging strand is _____ so that it passes through the polymerase active site in _____ direction, allowing synthesis to occur in the _____ direction.
Looped
Active site in 3' --\> 5'
Synthesis in 5' --\> 3'
80
DNA polymerase I is the _____ polymerase and deals with \_\_\_\_\_
Proofreading, deals with primer
81
The joining of Okazaki fragments by _____ and \_\_\_\_\_
DNA Polymerase I and DNA ligase
82
Problem: After Okazaki fragment synthesis is complete, there is a _____ between the end of Okazaki fragment and RNA primer.
Gap
83
Nick translation
DNA Polymerase I extends the Okazaki fragment while its 5--\>3 exonuclease activity removes the RNA primer.
84
Nick translation adds _____ where primer was which fixes gaps.
Base pair
85
What is required to rejoin the break?
DNA Ligase (only needed for lagging strand)
86
DNA Ligase solidifies _____ in DNA and causes DNA to become _____ now.
Nicks in DNA
Continuous DNA now
87
What allows for removal of mismatched **bases**?
Exonuclease
88
3'--\>5' exonuclease activity contributes to the remarkably high fidelity of DNA replication - less than \_\_\_\_.
10^-8 errors/BP
89
Most uncorrected replication errors are subsequently corrected by....
The DNA repair enzymes (NOT proofreading)
90
Mistake rate is approximately...
1/100,000 bps, but DNA pol III and repair enzymes fix many
91
Human genome?
3.2 x 10^9 billion
92
Fruit fly large chromosomes
10x larger than E. coli, more complex (Millions of base pairs)
93
Human genome BPs?
6 billion
94
Eukaryotic chromosomes are \_\_\_\_\_.
Large linear, double-stranded DNA molecules
95
Prokaryotes have _____ DNA.
Circular
96
\_\_\_\_\_ of replication are required with each origin of replication representing a unit or replicon.
Multiple origins
97
Humans have how many replicons?
30,000 replicons
98
Proteins called _____ allow only one replication per replicon per round of DNA synthesis.
Licensing factors
99
DNA pol alpha is equivalent to _____ in prokaryotes
Pol I
100
Displays primase activity, initiates DNA replication in eukaryotes, generating a DNA molecule approx. 20 dNTP in length
DNA Pol alpha
Don't incorporate RNA
101
DNA poly delta: A more processive enzyme, extends the chains. The switch from alpha to delta is called \_\_\_\_\_
Polymerase switching
102
Unique structures at the ends of linear chromosomes =
Telomeres
103
Telomeres are at the _____ of DNA
Free end of linear DNA
104
Two problems telomeres have:
1. Susceptible to damage by nucleases
2. Due to nature of DNA synthesis, one strand will shorten upon each round of DNA synthesis
105
IF telomere is shortened?
BAD = cell death
106
What replicates telomeres and fixes the overhang/gap?
Telomerase
107
Telomerase is RNA-dependent \_\_\_\_\_
DNA pol
108
Telomerase contains...
An RNA template that it uses to extend the leading strand
109
In rapidly dividing cells, including cancer cells, telomeres must be maintained by the telomerase to \_\_\_\_\_, which would lead to cell death.
Prevent shortening
110
\_\_\_\_\_ activity is characteristic of cancer cells (replicate uncontrollably).
High-telomerase activity
111
Which is NOT required for DNA synthesis?
* deoxynucleotide triphosphates
* a primer
* a template strand
* a DNA polymerase
* endonucleases
Endonucleases
112
The 3’ to 5’ exonuclease activity of DNA polymerase III is critical for:
* the processivity of polymerization
* removal of RNA primers
* the high fidelity of polymerization
* dimerization of the polymerase subunits
* DNA polymerase III does not have a 3’ to 5’ exonuclease activity
The high fidelity of polymerization
113
Enzymes which use the power of ATP hydrolysis to separate DNA strands are called:
* Restriction endonucleases
* Topoisomerases
* Separases
* Helicases
* Primases
Helicases
114
You set up a DNA replication reaction in a test tube using enzymes from E. coli. You add all the necessary components EXCEPT that you forget to add **DNA ligase**. When you then separate the strands to analyze the new DNA, what products would you expect?
* no DNA product at all
* normal full length DNA products only
* a mixture of full length strands, and shorter strands that include both RNA and DNA
* a mixture of full length strands, and shorter strands made of just DNA
* only short strands, which are made of just DNA
A mixture of full length strands, and shorter strands made of just DNA
115
You try your reaction again, remembering to add DNA ligase. This time, however, you forget to add **primase**. What products would you expect?
* no DNA product at all
* normal full length DNA products only
* a mixture of full length strands, and shorter strands that include both RNA and DNA
* a mixture of full length strands, and shorter strands made of just DNA
* only short strands, which are made of just DNA
No DNA product at all
116
Processivity refers to the:
Ability to catalyze many reactions without releasing the substrate
117
Which statement about DNA replication in eukaryotes in FALSE? Eukaryotic DNA replication:
* involves multiple DNA polymerases
* occurs on linear double-stranded DNA molecules
* utilizes multiple origins of replication
* utilizes DNA primers made by primase
* is highly processive
Utilizes DNA primers made by primase
