Chapter 11 Flashcards
1
Q
What is DNA replication?
A
The process in which original DNA strands are used as templates for the synthesis of new DNA strands.
2
Q
DNA replication relies on what?
A
The complementarity of DNA strands according to the AT/GC rule.
3
Q
What are template strands?
A
In DNA replication, the DNA strand that is used as a template.
4
Q
What’s another name for template strand?
A
Parental strand
5
Q
After the double helix have separated, what happens?
A
Individual nucleotides have access to the template strands.
6
Q
Hydrogen bonding between individual nucleotides and the template strands must obey what?
A
The AT/GC rule
7
Q
How is the replication process complete?
A
A covalent bond is formed between the phosphate of one nucleotide and the sugar of the previous nucleotide.
8
Q
The two newly made strands are referred to as what?
A
Daughter strands.
9
Q
DNA replication results in both copies what?
A
Retaining the same information- the same base sequence- as the original molecule.
10
Q
What features of DNA structure enable it to be replicated?
A
Its double-stranded structure
The base pairing between A and T and between G and C
11
Q
What are the three different mechanisms to explain the net result of DNA replication?
A
Conservative Model
Semiconservative Model
Dispersive Model
12
Q
What is the conservative model?
A
An incorrect model in which both strands of parental DNA remain together following DNA replication.
13
Q
In the conservative model what is the result?
A
The original arrangment of parental DNA remain together while the two newly made daugther strands remain together.
14
Q
What is the semiconservative model?
A
The correct model for DNA replication in which the newly made double-stranded DNA contains one parental strand and one daugther strand.
15
Q
What is the result of the semiconservative model?
A
The newly made double-stranded DNA contains one parental strand and one daugther strand.
16
Q
What is the dispersive model?
A
An incorrect model for DNA replication in which segments of parental DNA and newly made DNA are interspersed in both strands following the replication proccess.
17
Q
What did Matthew Meselson and Franklin Stahl do as an experiment? When?
A
Devised a method to experimentally distinguish newly made daughter strands from the original parental strands in 1958
18
Q
What was Meselson and Stahl’s technique?
A
Labeling DNA with a heavy isotope of nitrogen.
19
Q
Why did Meselson and Stahl use nitrogen?
A
It is found in the bases of DNA in a heavy (15N) or light (14N) form.
20
Q
What did Meselson and Stahl do prior to their experiment?
A
They grew E coli cells in the presence of 15N for many generations. So all the DNA was heavy-labeled.
21
Q
At the start of Meselson’s and Stahl’s experiment what did they do?
A
They switched the bacteria to a medium that contained only 14N and then collected sample of cells after various time points.
22
Q
What happened during Meselson’s and Stahl’s experiement?
A
Under the growth conditions, the cells replicated their DNA and divided into daugther cells every 30 minutes. After each cell doubling, the new daugther cells were viewed. The new daughter cells were labeled with light nitrogen, while the original strands remained heavy.
23
Q
How did Meselson and Stahl analyze the density of the DNA?
A
By centrifugation, using a ceisum chloride gradient. If both strands were light they would be at the top. If both strands were heavy they would be at the bottom, if there was one of each they would be in the middle.
24
Q
After two rounds of DNA replication, what did Meselson and Stahl find?
A
A mixture of light DNA and Half-heavy DNA which was consistent with the semiconservative model.
25
The complementarity of DNA strands is based on
a. the chemical properties of a phosphodiester linkage
b. the binding of proteins to the DNA
c. the AT/GC rule.
d. none of the above.
c. the AT/GC rule.
26
To make a new DNA strand, which of the following is necessary?
a. a template strand
b. nucleotides
c. heavy nitrogen
d. both a and b.
d. both a and b.
27
The model that correctly describes the process of DNA replication is
a. the conservative model
b. the semiconservative model
c. the dispersive model.
d. all of the above.
b. the semiconservative model
28
Bacterial chromosomes have a single what?
Origin of replication.
29
The synthesis of new daughter strands is initiated how?
Within the origin and proceeds in both directions.
30
What is bidirectionally?
The phenomenon in which two replication forks move in opposite directions, outward from the origin.
31
What are replication forks?
The regions in which two DNA strands have separated and new strands are being synthesized.
32
Eventually the replication forks do what?
Meed each other on the opposite side of the bacterial chromosome to complete the replication process.
33
What is the name of the origin of replication in E coli?
oriC (origin of Chromosomal Replication)
34
What are the three types of DNA sequences found in oriC?
An AT-rich region
DnaA box sequence
GATC methylation sites.
35
The AT-rich region is composed of what?
Three similar sequences that are 13bp long.
36
What are the functions of the AT-rich region and DnaA boxes?
The DnaA boxes are recognized by DnaA protein, which binds to them and causes the DNA to unwind at the At-rich region.
37
DNA replication is initated by what in bacterial chromosomes?
DnaA protein
38
What are DnaA proteins?
Proteins that binds to the DnaA box sequence at the origin of replication in bacteria and initiates DNA replication.
39
What are DnaA box sequences?
Serves as a recognition site for the binding of the DnaA protein, which is involved in the initiation of bacterial DNA replication.
40
When DnaA proteins are in their ATP-bound form, what do they do?
Bind to the five DnaA boxes in oriC to initiate DNA replication.
41
DnaA proteins also do what?
Bind to each other to form a complex.
42
What are other DNA-binding proteins that help DnaA proteins?
HU
| IHF
43
What does DnaA result in?
It bends around the complex of DnaA proteins and results in the separation of the At-rich region.
44
Why is the AT-rich region easy to separate?
Because AT base pairs only have two hydrogen bonds while GC has three.
45
How many replication forks are formed at the origin?
Two
46
What happens after separation of the AT-rich region?
The DnaA proteins, with the help of DnaC proteins, recruit DNA helicase proteins.
47
What are DNA helicase proteins?
An enzyme that separates the two strands of DNA.
48
What is another name for DNA helicase?
DnaB protein
49
What happens when DNA helicase encounters a double-stranded region?
It breaks the hydrogen bonds between the two strans, thereby generating two single strands.
50
How do DNA helicases separate the two strands?
Two DNA helicases start at teh oriC region and continue to separate the DNA strands. They use the energy from ATP hydrolysis to catalyze the separation of the double-stranded parental DNA. DNA hlicases bind to single stranded DNA and travel along the DNA in a 5' to 3' direction to keep the replication fork moving.
It promotes the movement of two replication forks outward from oriC in opposite direction.
51
What is bidirectional replication?
The phenomenon in which two DNA replication forks emanate in both directions from an origin of replication.
52
The GATC methylation sites within oriC are involved in what?
Regulating DNA replication.
53
GATC sites are methylated by what?
An enzyme known as DNA adenine methyltransferase (Dam).
54
Prior to DNA replication, the GATC sites are what?
Methylated in both strands. Which facilitates the initiation of DNA replication at the origin.
55
Following DNA replication, the newly made strands are not what?
Methylated, because adenine rather than methyladenine is incorporated into the daughter strands.
56
The initiation of DNA replication at the origin does not readily occur until what?
After it has become fully methylated.
57
Why does DNA replication not occur again too quickly?
It takes several minutes for Dam to methylate the GATC sites within the region.
58
A site in a chromosome where DNA replication begins is
a. a promoter
b. an origin of replication
c. an operator
d. a replication fork.
b. an origin of replication
59
The origin of replication in E. coli contains
a. an AT-rich region
b. DnaA box sequences
c. GATC methylation sites.
d. all of the above.
d. all of the above.
60
What is the function of DnaA proteins?
Bind to DnaA box sequences within the origin to initiate DNA replication
61
What is the function of DnaC proteins?
Aid DnaA in the recruitment of DNA helicase to the origin.
62
What is the function of DNA helicase (DnaB)
Separates the double-stranded DNA
63
What is the function of Topoisomerase II (DNA gyrase)?
Removes positive supercoiling ahead of the replication fork
64
What is single-strand binding proteins?
Bind to single-stranded DNA and prevent it from re-forming a double-stranded structure.
65
What is the function of Primase?
Synthesizes short RNA primers.
66
What is the function of DNA polymerase III?
Synthesizes DNA in the leading and lagging strands.
67
What is the function of DNA polymerase I?
Removes RNA primers, fills in gaps with DNA
68
What is the function of DNA ligase?
Covalently attaches adjacent Okazaki fragments.
69
What is the function of Tus?
Binds to ter sequences and prevents the advancement of the replication fork.
70
Why is primase needed for DNA replication?
DNA polymerase cannot initiate DNA replication on a bare template strand.
71
How does unwinding of the helix occur?
DNA helicase breaks the hydrogen bonds between the base pairs and thereby unwinds the strands. This action generates positive supercoiling ahead of each replication fork. Topoisomerase II travels in front of DNA helicase and alleviates positive supercoiling.
72
After the two parental DNA strands have been separated and the supercoiling relaxed, what must happen?
The strands must be kept separated until the complementary daughter strands have been made. This is done by single-strand binding proteins. They bind to the strands of parental DNA and prevent them from re-forming a double helix. The bases within the parental strands are kept in an exposed condition that enables them to hydrogen bond with individual nucleotides.
73
What happens during DNA replication?
Starts with the synthesis of short strands of RNA called RNA primers. These strands of RNA are synthesized by the linkage of ribonucleotides via an enzyme known as primase. This enzyme synthesizes short strands of RNA usually 10-12 nucleotides, and these strands prime the process of DNA replication.
74
What is the leading strand?
A strand during DNA replication that is synthesized continusouly toward the replication fork.
75
What is the lagging strand?
A strand during DNA replication that is synthesized as short Okazaki fragments in the direction away from the replication fork.
76
What happens in the leading strand?
A single primer is made at the origin of replication.
77
What happens in the lagging strand?
Multiple primers are made.
78
What happens during the synthesis of DNA?
DNA polymerase catalyzes the formation of covalent bonds between adjacent nucleotides and thereby makes the new daugther strands.
79
In E coli, what five proteins function as DNA polymerase?
```
Polymerase I
Polymerase II
Polymerase II
Polymerase IV
Polymerase V
```
80
In E coli, Polymerases I and III are involved in what?
Normal DNA replication
81
In E coli, Polymerases II, IV and V are involved in what?
DNA repair and replication of damaged DNA.
82
DNA polymerase III is responsible for what?
Most of the DNA replication because it is a large enzyme consisting of 10 different subunits that play various roles in the DNA replication process.
83
The complex of all 10 subunits of Polymerase III is called what?
DNA polymerase III holoenzyme
84
What are the subunits of Polymerase III?
```
Alpha
Epsilon
Theta
Beta
Tau, gamma, delta, delta', Psi, Chi
```
85
What is the function of the subunit alpha in polymerase III?
Catalyses the bond formation between adjacent nucleotides and synthesizes DNA
86
What is the function of the subunit Epsilon in polymerase III?
3' to 5' proofreading (removes mismatched nucleotides).
87
What is the function of the subunit theta in polymerase III?
Accessory protein that stimulates the proofreading function.
88
What is the function of the subunit beta in polymerase III?
Clamp protein, which allows DNA polymerase to slide along the DNA without falling off.
89
What are the functions of the subunits Tau, gamma, Delta, Delta', Psi, Chi in polymerase III?
Clamp loader complex, involved with helping the clamp protein bind to the DNA.
90
What are the common structural features of DNA polymerases in both bacterial species?
The catalytic subunit of all DNA polymerases has a strcutre resembling a human hand.
91
What common procedure is seen with DNA polymerases in all bacterial species?
The template DNA is threaded through the palm of the hand; the thumb and fingers are wrapped around the DNA. The incoming deoxyribonucleoside triphosphates (dNTPs) enter the catalytic site, bind to the template strand according to the AT/GC rule, and then are covalently attached to the 3' end of the growing strand. DNA polymerase also contains a 3' exonuclease site that removes mismatched bases.
92
Is the template strand read in the 5' to 3' or the 3' to 5' direction?
3' to 5'
93
What are the two unusual features of DNA polymerase?
It cannot begin DNA synthesis by linking together the first two individual nucleotides. Rather the enzyme can elongate only a preexisting strand starting with an RNA primer or existing DNA strand.
Second, the directionality of strand synthesis can only occur in the 5' to 3' direction.
94
What is the result of the two unusual features of Polymerase III?
The synthesis of the leading and lagging strands show distinctive differences.
95
How does the synthesis of the leading strand occur?
DNA polymerase III catalyzes the attachment of nucleotides to the 3' end of each primer, in a 5' to 3' direction. One RNA primer is made at the origin, and then DNA polymerase III attaches nucleotides in a 5' to 3' direction as it slides twoard the opening of the replication fork. The synthesis of the leading strand is continuous.
96
How does the synthesis of the lagging strand occur?
The synthesis of DNA also elongates in a 5' to 3' manner, but it does so in the direction away from the replication fork. RNA primers repeatedly initiate the synthesis of short segments of DNA; the synthesis is discontinuous.
97
In bacteria, how long are the fragments added on the lagging strand?
1000-2000 nucleotides.
98
In eukaryotes, how long are the fragments added on the lagging strand?
100-200 nucleotides.
99
Each fragment that is added to the lagging side contains what?
A short RNA primer at the 5' end, which is made by primase. The remainder of the fragment is a strand of DNA made by DNA polymerase and are known as Okazaki fragments.
100
Why are they called Okazaki fragments?
They are named after Reigi and Tuneko Okazaki who discovered them in 1960s.
101
What events must occur to complete the synthesis of the Okazaki fragments?
Removal of the RNA primers,
Synthesis of DNA in the area where the primers have been removed
The covalent attachment of adjacent fragments of DNA.
102
In E. coli, how are RNA primers removed?
By the action of DNA polymerase I. The enzyme has a 5' to 3' exonulcease activity, which means that it digests away the RNA primers in a 5' to 3' direction leaving a vacant area. DNA polymerase I then synthesizes DNA to fill in this region and uses the 3' end of an adjacent Okazaki fragment as a primer. So it would remove RNA primer from the first Okazaki fragment and synthesize DNA in the vacant region by attaching nucleotides to the 3' end of the second Okazaki fragment.
103
What does DNA ligase do again?
It catalyzes a covalent bond between adjacent Okazaki fragments to complete the replication process in the lagging strand.
104
In E coli, what does DNA ligase require?
NAD+ to carry out the reaction
105
In eukaryotes and archaea, what does DNA ligase require?
ATP
106
What is primosome?
A multiprotein complex composed of DNA helicase, primase, and several accessory proteins.
107
Primosome does what?
Leads the way at the replication fork. It tracks along the DNA, separating the parental strands and synthesizing RNA primers at regular intervals along the lagging strand. By acting within a complex, the actions of DNA helicase and primase can be better coordinated.
108
What is a replisome?
A complex that contains a primosome and dimeric DNA polymerase.
109
What is dimeric DNA polymerase?
A complex of two DNA polymerase proteins that move as a unit during DNA replication.
110
For Dimeric DNA polymerase to occur what must happen?
The lagging strand is looped out with respect to the DNA polymerase that synthesizes the lagging strand.
111
The loop allows what?
The lagging-strand polymerase to make DNA in a 5' to 3' direction yet move toward the opening of the replication fork.
112
When the DNA polymerase part of the Dimeric DNA polymerase reaches the end of the Okazaki fragment what happens?
It must be released from the template DNA and hop to the RNA primer that is closest to the fork.
113
The clamp loader complex does what?
Reloads the holoenzyme at the site where the next RNA primer has been made.
114
The clamp loader complex is part of what?
DNA polymerase holoenzyme
115
What are termination sequences?
In E. coli, a pair of sequences in the chromosome that bind a protein known as the termination utilization substance (Tus), which stops the movement of the replication forks.
116
What does Tus stand for?
Termination Utilization substance.
117
One of the ter sequences, designated T1 does what?
Prevents the advancement of the fork moving left to right, but allows the movement of the other fork.
118
What does T2 do?
Prevents the advancement of the fork moving right to left, but allows the advancement of the other fork.
119
In any given cell, only one what is required to stop the advancement of a fork?
Only one ter sequence because the other fork ends its synthesis of DNA when it reaches the halted replication fork.
120
What happens after the two forks stop?
DNA ligase covalently links the two daugther strands, creating two circular double stranded molecules.
121
DNA replication of bacteria often results in what?
Two intertwined DNA molecules known as catenanes.
122
What are catenanes?
Interlocked circular molecules.
123
In E. coli, how are catenanes unlocked?
Topoisomerase II introduces a temporary break into the DNA strands and then rejoins them after the strands have become unlocked.
124
Is DNA strand breakage neede for catenane separation?
Yes, so the strands can move relative to each other and untangle
125
How were most of the proteins involved in DNA replication discovered?
Isolation of mutants in E. coli that have abnormalities in DNA replication.
126
Who discovered the first DNA replication mutant? When? What was it?
Paula DeLucia and John Cairns in 1969. The gene that encodes DNA polymerase I.
127
What are conditional mutants?
A mutant whose phenotype depends on the environment conditions, such as temperature-sensitive mutant.
128
What is temperature sensitive (ts) mutant?
A mutant that has normal phenotype at a permissive temperature, but a different phenotype, such as failure to grow, at the nonpermissive temperature.
129
What is a general strategy to isolate ts mutants?
Expose bacterial cells to a mutagen that increase the likelihood of mutations.
The mutagenized cells are plated on growth media and incubated at the permissive temperature. The colonies are then replica plated onto two plates: one incubated at the permissive temperature and one at the nonpermissive temperature. Thus they can identify ts mutations
130
What are rapid-stop mutations?
Mutations found in genes that encode proteins needed for DNA synthesis, and thus showed rapid arrest of DNA synthesis.
131
What are slow-stop mutants?
Involved genes that encode proteins needed for the intiation of replication at the origin. They were able to complete their current round of replication, but could not start another round.
132
What are some Rapid-stop mutants?
```
dnaE
dnaX
dnaN
dnaZ
dnaG
dnaB
```
133
What is the function of dnaE?
alpha subunit of DNA polymerase III, synthesizes DNA.
134
what is the function of dnaX?
Tau subunit of DNA polymerase III; part of the clamp loader complex and also promotes the dimerization of two DNA polymerase III proteins together at the replication fork.
135
What is the function of dnaN?
Beta subunit of DNA polymerase III; functions as a clamp protein that makes DNA polymerase a processive enzyme
136
What is the function of dnaZ?
Gamma subunit of DNA polymerase III; helps the beta subunit bind to the DNA.
137
What is the function of dnaG?
Primase; needed to make RNA primers.
138
What is the function of dnaB?
Helicase; needed to unwind the DNA strands during replication.
139
What are thw slow-stop mutants?
dnaA
| dnaC
140
What is the function of dnaA?
dnaA protein that recognizes the DnaA boxes at the origin
141
What is the function of dnaC?
DnaC protein that recruits DNA helicase to the origin.
142
The enzyme known as _________ uses ________ and separates the DNA strands at the replication fork.
a. helicase, ATP
b. helicase, GTP
c. gyrase, ATP
d. gyrase, GTP
a. helicase, ATP
143
In the lagging strand, DNA is made in the direction __________ the replication fork and is made as _________.
a. toward, one continuous strand
b. away from, one continueous strand.
c. toward, Okazaki fragments
d. away from, Okazaki fragments.
d. away from, Okazaki fragments.
144
DNA polymerases catalyze the ______________ between the ____________ and the ____________.
Covalent attachment between the phosphate in one nucleotide and the sugar in the previous nucleotide.
145
Prior to bond formation, the nucleotide about to be attached to the growing strand is a what?
Deoxyribonucleoside triphosphate (dNTP). It contains three phsphate groups attached at the 5'-C atom of deoxyribose.
146
dNTP first enters where and does what?
The catalytic site of DNA polymerase and binds to the template strand according to the AT/GC rule.
147
What happens after dNTP enters the catalytic site?
The 3'-OH group on the previous nucleotide reacts with the phosphate group adjacent to the sugar on the incoming nucleotide. This reaction is highly exergonic and results in a covalent bond between the sugar at the 3' end of the DNA strand and the PO4 2- of the incoming nucleotide.
148
The formation of the covalent bond between the phosphate and sugar causes what?
The newly made strand to grow in the 5' to 3' direction. Pyrophosphate (PPi) is released and broken down into 2 phosphates.
149
Does the oxygen in the newly made ester bond come from the phosphate or from the sugar?
The sugar
150
In E. coli, DNA polymerase III attaches how many nucleotides per second?
750
151
What is a processive enzyme?
An enzyme, such as RNA and DNA polymerase, which glides along the DNA and does not dissociate from the template strand as it catalyzes the covalent attachment of nucleotides.
152
What is an example of a processive enzyme?
DNA polymerase III.
153
What subunit makes DNA polymerase III so fast? Why?
Beta subunit. If forms a dimer in the shape of a ring; the hold of the ring is large enough to accommodate a double-stranded DNA molecule. and its width is about one turn of DNA.
154
The absense of the beta subunit results in synthesis of how many nucleotides a second?
ONly 20 and may result in the polymerase falling off the DNA template after 10 nucleotides.
155
How many mistakes occur with DNA polymerase per 100 million nucleotides?
1
156
What is fidelity?
A term used to describe the accuracy of a process. If there are few mistakes, a preocess has high fidelity.
157
What are the three mechanisms to ensure fidelity of DNA replication?
Stability of Base Pairing
Structure of the active site of DNA polymerase
Proofreading.
158
What is stability of base pairing?
The hydrogen bonding between G and C or A and T is much more stable than between mismatched pairs, so only 1 mistake per 1000 nucleotides would be made.
159
What is structure of the active site of DNA polymerase?
The active site preferentially catalyzes the attachment of nucleotides when the correct bases are located in opposite strands. Helix distortions caused by mispairing usually prevent an incorrect nucleotide from properly occupying the active site of DNA polymerase. The correct nucleotide occupies the active site with precision and promotes induced fit, which is a conformational change in the enzyme that is necessary for catalysis. So the error rate is 1 in 100,000 to 1 million.
160
What is proofreading?
DNA polymerase enzymatically removes mismatched nucleotides. This occurs by exonuclease cleavage of the bonds between adjacent nucleotides at the 3' end of the newly made strand, called the proofreading function. Proofreading occurs by the removal of nucleotides in the 3' to 5' direction at the 3' exonuclease site. After the mismatched nucleotide is removed, DNA polymerase resumes DNA synthesis in the 5' to 3' direction.
161
What is the proofreading function?
The ability of DNA polymerase to remove mismatched bases from a newly made strand.
162
DNA polymerase III is a processive enzyme, which means that
a. it does not dissociate from the growing strand after it has attached a nucleotide to the 3' end.
b. it makes a new strand very quickly
c. it proceeds toward the opening of the replication fork.
d. it copies DNA with relatively few errors.
a. it does not dissociate from the growing strand after it has attached a nucleotide to the 3' end.
163
The proofreading function of DNA polymerase involves the recognition of a _________ and the removal of a short segment of DNA in the _______direction.
a. missing base, 5' to 3' direction
b. base mismatch, 5' to 3'
c. missing base, 3' to 5'
d. base mismatch, 3' to 5'
d. base mismatch, 3' to 5'
164
What are the similarities between prokaryote and eukaryote DNA replication?
DNA helicases, topoisomerases, single-strand binding proteins, primases, DNA polymerases, and DNA ligases have been identified in eukaryotes.
165
How is prokaryote DNA replication and Eukaryote DNA replication different?
Eukaryotic DNA replication is more complex.
166
How does DNA replication initiation occur in linear eukaryotic chromosomes.
At Multiple origins of replication.
167
How provided evidence of multiple origins of replication and when?
Joel Huberman and Arthur Riggs in 1969
168
How did Huberman and Riggs demonstrate the multiple origins?
By adding a radiolabeled nucleoside to a culture of actively dividing cells, followed by a chase with nonlabeled deoxythymidine. The radiolabeled deoxythymidine was taken up by the cells and incorporated into their newly made DNA strands for a brief period. The chromosomes were then isolated fro the cells and subjected to autoradiography. The radiolabeled segments were interspersed among the nonlabeled segments, thus showing multiple origins of replication.
169
DNA replication proceeds how?
Bidirectionally from many origins of replication.
170
DNA replication occurs during which phase of the cell cycle?
S phase
171
The multiple replication forks eventually do what?
Make contact with each other to complete the replication process.
172
WHy do eukaryotes need multiple origins of replication?
They are so large, they needs multiple origins so the DNA can be replicated in a reasonable length of time.
173
What are ARS elements?
DNA sequences found in yeast that function as origins of replication.
174
What does ARS stand for?
Autonomously replicating sequences.
175
What is the length of ARS elements?
50bp
176
ARS contain what?
A higher percentage of A and T bases.
| They also have a copy of the ARS consensus sequence and other elements that enhance origin function.
177
What is the ARS consensus sequence?
ATTTAT(A or G)TTTA
178
In animals, origins are not determined by what?
Particular DNA sequences, but instead occur at specific sites along a chromosome due to features of chromatin structure such as histone modifications.
179
DNA replication in eukaryotes begins with the assembly of what?
Prereplication complex (preRC)
180
What is prereplication complex?
An assembly of at least 14 different proteins, including a group of 6 proteins called the origin recognition complex (ORC), that acts as the initiator of eukaryotic DNA replication.
181
What is the origin recognition complex (ORC)?
A complex of six proteins in eukaryotes that is necessary to initiate DNA replication.
182
ORC first bind to what?
The origin of replication and then other proteins of the preRC bind, including a group of proteins called MCM helicase.
183
What is MCM helicase?
A group of eukaryotic proteins needed to complete a process called DNA replication licensing, which is necessary for the formation of two replication forks at an origin of replication.
184
What is DNA replication licensing?
In eukaryotes, occurs when MCM helicase is bound at an origin, enabling the formation of two replication forks.
185
How many DNA polymerases do mammalian cells have?
Over a dozen.
186
What is the function of subunit alpha in eukaryote polymerase?
Initaties DNA replication in conjuction with Primase
187
What is the function of subunit epsilon in eukaryote polymerase?
Replication of the leading strand
188
What is the function of subunit delta in eukaryote polymerase?
Replication of the lagging strand.
189
What is the function of subunit gamma in eukaryote polymerase?
Replication of mitochondrial DNA
190
What is the function of subunit eta, kappa, iota, xi in eukaryote polymerase?
They are lesion-replicating polymerases and help with the Replication of damaged DNA
191
What are the functions of alpha, beta, delta, epsilon, sigma, lambda, mu, phi, theta, eta in eukaryote polymerases?
DNA repair or other functions. They have duel functions.
192
Which DNA polymerase subunit that is the only eukaryotic polymerase that associates with primase?
Alpha
193
What is the functional role of the DNA polymerase a/primase complex?
To synthesize a short RNA-DNA primer of approximately 10 RNA nucleotides followed by 20-30 DNA nucleotides.
194
The short RNA-DNA strand is used by what for what?
DNA polymerase epsilon or delta for the processive elongation of the DNA strands.
195
For the processive elongation of the DNA strand to occur, what must happen?
The DNA polymerase a/primase complex dissociates from the replication fork and is exchanged for DNA polymerase epsilon or delta.
196
What is polymerase switch?
During DNA replication, when one type of DNA polymerase is switched for another type.
197
What happens when DNA polymerase alpha, delta and epsilon encounter abnormalities in DNA structure, such as abnormal bases or crosslinks?
They replicate over the aberration which attracts lesion-replicating polymerases that have special properties that enable them to synthesize a complementary strand over the abnormal region. The different types of lesion-replicating polymerases may be able to replicate over different kinds of DNA damage.
198
What is another key difference between bacterial and eukaryotic DNA replication?
The way that RNA primers are removed. Bacterial RNA primers are remobed by DNA polymerase I. DNA polymerase enzyme does not play a role in eukaryotes. Instead an enzyme called flap endonuclease is responsible for RNA primer removal.
199
Where does flap endonuclease get its name?
From the fact that it removes small pieces of RNA flaps that are generated by the action of DNA polymerase delta.
200
How is a RNA flap generated?
DNA polymerase delta elongates the left okazaki fragment until it runs into the RNA primer of the adjacent Okazaki fragment on the right. This causes a portion of the RNA primer to form a short flap. As DNA polymerase delta continue to elongate the DNA, short flaps continue to be generated.
201
What happens when all the RNA primer is removed?
DNA ligase seals the DNA fragments together.
202
Can flap endonuclease remove a long flap?
No, long flaps must be cleaved by an enzyme called Dna2 nuclease/helicase.
203
What is telomeres?
Specialized DNA sequences found at the ends of linear eukaryotic chromosomes.
204
Telomeres consist of what?
Tandemly repeated sequences and a 3' overhang region that is 12-16 nucleotides in length.
205
What is a common feature of telomeres in a variety of eukaryotic organisms?
The telomeric sequence contains several guanine nucleotides and often many thymine nucleotides.
206
What is the telomeric repeat sequence of Mammals such as humans?
TTAGGG
207
What is the telomeric repeat sequence of slime molds such as physarum, didymium, and dictyostelium?
TTAGGG
| AG
208
What is the telomeric repeat sequence of filamentous fungi, such as neurospora?
TTAGGG
209
What is the telomeric repeat sequence of budding yeast, such as saccharomyces cerevisiae?
TG
210
What is the telomeric repeat sequence of ciliates?
Tetrahymena: TTGGGG
Paramecium: TTGGG(T/G)
Euplotes: TTTTGGGG
211
What is the telomeric repeat sequence of flowering platns, such as arabidopsis?
TTTAGGG
212
Why are telomeric repeat sequences needed?
Because DNA polymerase is unable to replicate the 3' ends of DNA strands.
213
Why is DNA polymerase unable to replicate the 3' ends of DNA strands?
Because of the two unusual features of the enzyme. It can only synthesize DNA in the 5' to 3' direction and it cannot link together the first two individual nucleotides, only elongate preexisting strands.
214
What would be the result if the 3' ends were never replicated?
They DNA strand would progressively become shorter.
215
How is the loss of genetic information due to chromosome shortening prevented?
Additional DNA sequences are attached to the ends of telomeres.
216
What did Carol Greider and Elizabeth Blackburn discover in 1984?
The enzyme telomerase
217
What does telomerase do?
prevent chromosome shortening by recognizeing the sequences at the ends of eukaryotic chromosomes and synthesizes additional repeats of telomeric sequences.
218
How many times would telomerase have to bind to a different site in the telomere to make a segment of DNA that is 36 nucleotides in length?
6 times
| 36 divided by 6
219
Telomerase contains what?
Both protein subunits and RNA. The RNA part of telomerase contains a sequence complementary to the DNA sequence found in the telomeric repeat.
220
What allows telomerase to bind to the 3' overhange region of the telomere?
The RNA part
221
What are the three steps for the replication of the ends by telomerase?
Binding of telomerase
Polymerization
Translocation.
222
What is polymerization?
Following binding, the RNA sequence beyond the binding site functions as a template for the synthesis of a 6 nucleotide sequence. It is called polymerization because it is analogous to the function of DNA polymerase.
223
Telomere lengthening is catalyzed by what?
Two identical protein subunits of telomerase called telomerase reverse transcriptase (TERT)
224
What is telomerase reverse transcriptase?
An enzyme within telomerase that uses RNA as a template to make DNA.
225
What is translocation?
Following polymerization, telomerase then moves to the new end of this DNA strand and attaches another 6 nuclotides to the end. This is called translocation .
226
What occurs many times in a row? What is the result?
The binding-polymerization-translocation cycle which lengthens the 3' end of the DNA strand in the telomeric region.
227
The complementary strand of DNA in eukaryotes is synthesized by what?
Primase, DNA polymerase, and DNA ligase. The RNA primer is later removed which leave a 3' overhang.
228
What tends to shorten with age?
Telomeres
229
At birth telomeres are how long, and in an elderly person telomeres are how long?
8,000bp
| 1500bp
230
What happens when telomerase becomes too short?
The cells become senescent
231
What is senescent?
A cell that is no longer capable of dividing.
232
What was Andrea Bodnar experiment and what did they find?
Inserted a gene that encodes a highly active telomerase into human cells. The expression of telomerase prevented telomere shortening and senescence.
233
How do cancer cells keep dividing if telomeres shorten at each cell division?
Cancer cells carry mutations that increase the activity of telomerase, thereby preventing telomere shortening.
234
In eukaryotes, DNA replication is intitiated at an origin of replication by
a. DnaA proteins
b. the origin recognition complex.
c. DNA polymerase delta
d. MCM helicase
b. the origin recognition complex.
235
Which of the following statements regarding DNA polymerases in eukaryotes is not correct?
a. DNA polymerase alpha synthesizes a short RNA-DNA primer.
b. DNA polymerases epsilon and delta synthesize most of the leading and lagging, respectively.
c. lesion-replicating DNA polymerases can replicate over damaged DNA.
d. all of the above statements are correct.
d. all of the above statements are correct
236
In eukaryotes, RNA primers are primarily removed by
a. DNA polymerase I
b. DNA polymerase alpha
c. flap endonuclease
d. helicase.
c. flap endonuclease
237
To synthesize DNA, what does telomerase use as a template?
a. it uses the DNA in the 3' overhang
b. it uses RNA that is a component of telomerase.
c. no template is used
d. both a and b are correct.
b. it uses RNA that is a component of telomerase.
238
With regard to DNA replication, define the term bidirectional replication.
DNA replication in both directions starting from one origin.
239
The chromosome of E coli contains 4.6 million bp. How long will it take to replicate its DNA? Assuming DNA polymerase III is the primary enzyme involved and this enzyme can actively proofread during DNA synthesis, how many base pair mistakes will be made in one round of DNA replication in a bacterial poplation containing 1000 bacteria?
4,600,000/750=6133 seconds or 102.2 minutes.
Because replication is bidirectional, 102.2/2= 51.1 minutes.
An error rate of one mistake per 100,000,000 nucleotides. then
4,600,000 X 1000 bacteria- 4,600,000,000 nucleotide replicated DNA
4,600,000,000/100,000,000=46 mistakes.
240
A DNA strand has the following sequence: 5'-GATCCCGATCCGCATACATTTACCAGATCACCACC-3'
In which direction would DNA polymerase slide along this strand (from left to right or from right to left)? If this strand was used as a template by DNA polymerase, what would be the sequence of the newly made strand? Indicate the 5' and 3' ends of the newly made strand.
DNA polymerase would slide right to left.
| The new strand would be 3'-CTAGGGCTAGGCGTATGTAAATGGTCTAGTGGTGG-5'
241
Sometimes DNA polymerase makes a mistake, and the wrong nucleotide is added to the growing DNA strand. With regard to pyrimidines and purines, two general types of mistakes are possible. The addition of an incorrect pyrimidine instead of the correct pyrimidine (cytosine where thymine should be added) is called a transition. If a pyrimidine is incorrectly added to the growing strand instead of purine (adding cytosine where adenine should be added) this type of mistake is called a transversion. If a transition or transversion is not detected by DNA polymerase, a mutation is created that permanently changes the DNA sequence. Though both types of mutation are rare, transition mutations are more frequent than transversion mutation. Based on your understanding of DNA replication and DNA polymerase, offer three explanations why transition mutations are more common.
1. According to the AT/GC rule, a pyrimidine always hydrogen bonds with a purine. A transition involves a pyrimidine hydrogen bonding to a purine, but a transversion causes a purine to hydrogen bond with a purine or a pyrimidine to hydrogen bond with a pyrimidine. The structure of the double helix makes it much more difficult for this later type of hydrogen bond to occur.
2. The induced-fit phenomenon of the active site of DNa polymerase makes it unlikely for DNA polymerase to catalyze covalent bond formation if the wrong nucleotide is bound to the template strand. A transition mutation creates a somewhat bad interaction between the bases in opposite strands, but it is not as bad as the fit caused by a transverse mutation. In a transversion, a purine is opposite another purine, or a pyrimidine is opposite a pyrimidine. This is a very bad fit.
3. The proofreading function of DNA polymerase is able to detect and remove an incorrect nucleotide that has been incorporated into the growing strand. A transversion is going to cause a larger distortion in the structure of the double helix and make it more likley to be detected by the proofreading function.
242
Single-strand binding proteins keep the two parental strands of DNA separated from each other until DNA polymerase has an opportunity to replicate the strands. sugest how single-strand binding proteins keep the strands separated and yet do not impede the ability of DNA polymerase to replicate the strands.
Primase and DNA polymerase are able to knock the single-strand binding proteins off the template DNA.
243
Discuss the similarities and differences in the synthesis of DNA in the lagging and leading strands. What is the advantage of a primosome and a replisome as opposed to having all replication enzymes functioning independently of each other?
The leading strand is primed once, at the origin, and then DNA polymerase III synthesizes DNA continuously in the direction of the replication fork. In the lagging strand, many short pieces of DNA are made. This requires many RNA primers. The primers are removed by DNA polymerase I, which then fills in the gaps with DNA. DNa ligase then covalently connects the Okazaki fragments. Having the enzymes within a complex such as a primosome or replisome provides coordination among the different steps in the replication process thereby allowing it to proceed faster and more efficiently.
244
What is the processive enzyme? Explain why this is an important feature of DNA polymerase.
A processive enzyme is one that remains clamped to one of its substrates. In the case of DNA polymerase, it remains clamped to the template strand as it makes a new daughter strand. This is important to ensure a fast rate of DNA synthesis.
