Unit 6 Flashcards
(120 cards)
1
Q
Which nucleotides are purines?
A
Adenine and Guanine
pure AS GOLD
2
Q
Which nucleotides are pyrimidines?
A
Cytosine Uracil and Thymine
3
Q
How can you recognize Adenine?
A
Two rings, NH2 at the top
4
Q
How to recognize Guanine?
A
Two rings, C=O at the top
5
Q
How to recognize Cytosine?
A
One ring, NH2
6
Q
How to recognize Thymine?
A
One ring, two carbonyls, one methyl
7
Q
How to recognize Uracil?
A
One ring, two carbonyls
8
Q
Draw the strucutre of ribose
A
Has an OH on the 2’ carbon
9
Q
Draw the structure of deoxyribose
A
Has an H on the 2’ carbon
10
Q
Given the structure of the bases in Fig 8-2 (p. 264) draw structures representing
a 2’-deoxynucleoside
A
Nucleoside means that you have a nitrogenous base and sugar group (sugar)
OH group on 1’ carbon is replaced by a nitrogenous base
11
Q
Given the structure of the bases in Fig 8-2 (p. 264) draw structures representing
a 2’-deoxynucleotide
A
Nucleotide means that you have the nitrogenous base, sugar group, and phosphate group added
Phosphate group replaces the OH in CH2OH
12
Q
Given the structure of the bases in Fig 8-2 (p. 264) draw structures representing
a 2’-deoxynucleoside 5’ -di- or triphosphate =
A
Add three phosphate groups
13
Q
Write out the numbering convention for the pentose group
A
14
Q
Which atom in the sugar is the base attached to? What kind of bond joins the sugar to the base?
A
The 1’ carbon
N-beta-glycosyl
15
Q
Identify the atom that is found at the 2’ position in deoxyribonucleotides? In ribonucleotides?
A
- Deoxy: two H’s
- Ribo: H on top, OH on bottom
16
Q
What functional group is at the 3’ position in ribo and deoxyribonucleotides? To which atom in the sugar is the phosphate bond?
A
- Hydroxyl group
- The 5’ carbon
17
Q
Discuss the nucleotides found in DNA. Compare and contrast these nucleotides to those found in RNA
A
DNA nucleotides: A,C,T,G
RNA nucleotides: A,C,U,G
18
Q
When we polymerize nucleotides, how many phosphate groups does the dTNP have to have?
A
has to be triphosphate
19
Q
Draw the reaction mechanism for polymerization of nucleotides:
A
- Polymerization is from 5’ to 3’ (because we are adding with the 3’ hydroxyl group)
- OH on 3’ carbon attacks the first phosphate group on the next nucleotide, pushing the electrons off of the oxygen adjacent to the phosphate.
- hydroxyl’s H is lost in the process of creating the phosphodiester bond
20
Q
What is the product of nucleotide polymerization?
A
pyrophosphate
21
Q
Draw out a section of DNA (use the letters A,C,G, and T to represent the structure of the base). Point out the following:
a. phosphodiester linkage
b. sugar-phosphate backbone
c. 5’ and 3’ ends
d. overall charge
A
- when you draw this, make sure your 5’ phosphate is pointed towards the ceiling and label it as 5’
- make sure every P has four O’s around it
a. binds the 3’ carbon of one pentose to the 5’ of another through phosphodiester linkage
b. alternativing pentose and phosphate bonds
c. always being added to the 3’ end, towards the bottom
d. negative due to the phosphate groups being ionized and having a negative charge at pH 7
22
Q
Distinguish between an oligonucleotide and polynucleotide ?
A
Oligonucleotide: short nucleic acid (usually fewer than 50 bp)
Polynucleotide: A longer nucleic acid
23
Q
Is the DNA helix right or left handed?
A
Right-handed
24
Q
What two factors keep strands together (pay attention to the phrasing of this question)?
A
- Hydrogen bonds between complementary bases
- Base stacking interactions (hydrophobic interactions)
25
Which bonds are stronger, G-C or A-T?
G-C bonds because they have 2 H=Bonds while A-T have two
We know this because when you try to separate DNA with more G-C pairs than A-T, it's harder to pull apart
26
Are A-T pairs or G-C pairs longer?
A-T pairs are longer (11.1 Angstroms) than G-C pairs (10.8 Angstroms)
But since purine will always be paired with a pyrimidine, the width/diameter of the double helix will remain the same
27
Where are the sugar phosphate backbones located on the DNA molecule?
They are located on the outside facing the water because the phosphate contains a negative charge. That means it is hydrophilic and wants to interact with water
28
Where are the stacked base pairs located? Why?
Bases are aromatic and nonpolar. Therefore, they are hydrophobic and do not interact with water. Due to the hydrophobic effect, the forces between the bases is stronger.
29
Major and Minor group (draw this out)
The offset pairing of the two strands creates a major and minor groove. The major groove has a bigger space between the strands while the minor strand has a smaller space
30
What word is used to describe the polarity of the two strands?
They are antiparallel
31
What two forces STABILIZE the DNA double helix?
***
Technically there are three
- Metal cations: The double helix is primarily stabilized by metal cations that shield the negative charges of the backbone phosphates
- Base stacking interaction between successive base pairs. GC bonds are stronger than the successive A-T pairs
- The hydrogen bonds do not contribute significantly to the stability of the structure
32
How can DNA be reversibly denatured?
When subjected to extreme pH's or temperatures above 80ºC
33
Discuss the types of bonds broken during denaturation?
-Hydrogen bonds and the base-stacking interactions (hydrophobic interaction) causes unwinding of the double helix to form two single strands
- No covalent bonds are broken
34
Describe the experimental procedures for generating this graph. How is percent denaturation quantified?
Draw out what the x and y axis are
***
The DNA's temperature increases at controlled rates and the concentration of double stranded DNA is checked with fluorescence
35
What is the relationship between tm and the base composition? Why does this relationship exists?
- LINEAR RELATIONSHIP. The higher the content of G-C base pairs, the higher the melting point of the DNA. This is because G-C pairs make greater contributions to stacking than do A-T pairs
35
What is the melting point (tm)
The temperature at which 50% of the DNA strands are presented as single separated strands
36
Define annealing
When the temperature or pH is returned to the range in which most organisms live, the unwound segments of the two strands spontaneously rewind, or anneal, to yield the intact duplex
37
Do Worked Example 8-1 on Base Pairing in DNA
38
Define and distinguish between DNA replication, transcription, and translation
- DNA Replication: copying of parental DNA to form daughter DNA with identical nucleotide sequences
- Transcription: parts of the genetic message encoded in DNA are copied precisely into RNA
- Translation: genetic message encoded in messenger RNA is translated on the ribosomes into a polypeptide with a particular sequence of amino acids
39
What is a gene?
All the DNA that encodes the primary sequence of some final gene product, which can be either a polypeptide or an RNA with a structural or catalytic function
40
What distinguishes a plasmid from a chromosome?
- Chromosome: A single large DNA molecule and its associated proteins and often associated regulatory or structural RNA; containing many genes; stores and transmits genetic information
- Plasmids: in addition to the very large, circular DNA chromosomes in their nucleoid, many bacteria contain one or more small circular DNA molecules that are free in the cytosol.
--> many plasmids have no obvious advantage to their host and seem to have self-propagation as their only function. However, some plasmids carry genes that are useful to host bacterium (like making bacteria resistant to antibacterial agents)
41
Is all eukaryotic DNA stores in the nucleus?
No, there is some DNA that can be found in the mitochondria and chloroplast due to the endosymbiont theory
42
Intron
A sequence of nucleotides that is transcribed but removed from the RNA transcript before translation; also called intervening sequence
43
Exon
"coded region". The segment of a eukaryotic gene that encodes a portion of the final product of the gene; a segment of RNA that remains after post-trasnlational processing and is transcribed into a protein or incorporated into the structure of an RNA
44
Satellite DNA
Also referred to as simple-sequence DNA. Highly repeated, non translational segments of DNA in eukaryotic chromosomes; most often associated with the centromeric region. It's function is unknown and it is called satellite because it often separates from the rest of the DNA.
45
Centromere
A sequence of DNA that functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindle
46
Telomere
Sequences at the ends of eukaryotic chromosomes that help stabilize the chromosome. They end with multiple repeat sequences
47
How can the linking number change?
Only if you break one or both of the strands
48
What is the linking number of DNA in a relaxed state?
1 turn = 10.5 base pairs
Linking number = total # of base pairs/10.5
49
What is meant by linking number?
Number of times one strand wraps around the other strand
50
Calculated the linking number of a circular 315 bp DNA molecule that is in the fully relaxed state
Linking number = total # of base pairs/10.5
315/10.5 = 30
51
What is a topoisomer?
Two forms of circular DNA that differ only in a topological property such as linking number
52
What kind of property is linking number?
A topological property
53
Discuss how over-winding or underwinding the DNA double helix leads to the formation of positive or negative supercoils respectively
- By increasing or decreasing linking #, it introduces torsional strain on the helix
- Overwinding leads to positive super coils which are left handed
- Underwinding leads to negative super coils which are right handed
54
List two reasons why it is advantageous for cells to maintain their DNA in the underwound state?
1. Supercoils allow for DNA packaging (both underwinding dna overwinding can do this)
2. Underwinding facilitates strand separation. This means that there is space between the strands that can allow for proteins to come in and facilitate DNA replication
55
What is a topoisomerase?
Enzymes that introduce positive or negative supercoils in a closed, circular duplex
They change DNA linking number by breaking the strand and twisting it.
56
Differentiate between type I and type II topoisomerases in E.coli
- Type I topoisomerases act by transiently breaking one of the two DNA strands, passing the unbroken strand through the break and regioning the broken ends; increases the link number by increments of 1
- Type II topoisomerases break both DNA strands and change LN in increments of 2
57
Describe how E.coli topoisomerase I removes negative supercoils, thereby increasing the linking number
1. Active site Tyr attacks a phosphodiester bond in one DNA strand, cleaving it and creating a covalent 5'-phosphotyrosyl protein-DNA linkage
2. Enzyme changes to an open conformation
3. The unbroken DNA strand passes through the break in the first strand
4. Enzyme in closed conformation; liberated 3' -OH attacks the 5'-phosphotyrosyl protein DNA linkage to religate the cleaved DNA strand (picks up H from surrounding to go back to the Tyr)
58
Discuss the packaging of DNA into nucleosomes
- DNA chromatin closely associated to histone proteins -->which package and order the DNA into structural units called nucleosomes
- Nucleosomes --> structural units for packaging chromatin core, consists of a DNA strand wound around a histone core
59
What is unique about the amino acid composition of histone proteins that is crucial to their DNA-binding function?
- Histones are rich in Arginine and Lysine (+) which can tightly bind DNA which is negatively charged
60
Explain what is meant by semiconservative replication
Each DNA strand serves as a template for the new strand. Meaning that all DNA have an old strand and a new strand
61
Define what is meant by the term origin of replication
The nucleotide sequence or site in DNA where replication is initiated
62
What is a replication fork? Discuss bi-directional replication
- Replication fork: a y-shaped structure where new DNA strands are synthesized by a multienzyme complex
-->Parent DNA is unwound, separated strands quickly replicate
- Bidirectional: Both ends of the loop have active replication forks and the DNA strands are replicated simultaneously
63
What is the direction of DNA synthesis?
5' to 3'
64
What is the direction that DNA Polymerase reads the template strand?
3' to 5'
65
Which strand is replicated continuously, and which discontinuously?
The leading strand is replicated continuously and the lagging strand is discontinuous
66
What are Okazaki fragments?
Fragments of discontinuous DNA on the lagging strand
67
Does the leading strand move in the direction of the replication fork or in the opposite direction? What about the lagging strand?
- Leading moves in the same direction which is why it's synthesized in 5' to 3'
- Lagging is in the opposite direction and in fragments so that the DNA is still synthesized in 5' to 5'
68
Distinguish between an exonuclease and an endonuclease
- Exonuclease: degrade nucleic acids from one end of the molecule whether it is the 5' or 3' end of the DNA
- Endonuclease: can degrade at specific internal sites in a nucleic acid strand or molecule, reducing it to smaller and smaller fragments
69
What are the two "central requirements" of DNA polymerase for DNA polymerization?
1. All DNA polymerases require a template
2. The polymerases require a primer - strand segment with free 3'-OH group to which a nucleotide can be added
* In the leading strand, there only ever one primer added by a primase. The new primer after that is just the 3' -OH of the newly synthesized strand. But for the lagging strand, there are primases constantly adding primers
70
Which type of nucleotide (dNMP, dNDP, or dNTP) are used as substrates?
dNTP
71
What is the purpose of the Aspartate and Mg2+?
The negative charge from the Asp will coordinate the Mg2+. Mg2+ cations are important for positioning and orienting the negatively charged substrates during DNA synthesis
72
Explain the reaction between the substrates at the level of arrow pushing
- Negatively charged -OH group on primer attacks positively charged phosphate on incoming dTNP
73
What process drives the reaction towards the formation of products? Discuss in terms of Le Chatelier's Principle
- When pyrophosphate is generated, an enzyme hydrolyzes it into 2 inorganic phosphates
- The absence of pyrophosphate drives the reaction forward
DNA(n) +dNTP <---> DNA (n+1) + PPi (immediately hydrolyzed to 2 Pi)
74
What kind of bond is formed in the reaction catalyzed by DNA polymerases?
A phosphodiester bond
75
Define processivity
- The average number of nucleotides added before a polymerase dissociates
- After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide
76
Discuss how the geometry of the active site of DNA polymerase contributes to fidelity of DNA replication? What is another word for this site?
- Standard AT and GC pairs have similar geometries, an active site sized to fit one will generally accommodate the other
- Geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase (incorrect bases will not fit in active site)
*Another word for this site is the insertion site
77
Discuss proofreading by DNA Polymerase. Which exonuclease activity is responsible for DNA proofreading?
- One mechanism intrinsic to many DNA polymerases is a separate 3' to 5' exonuclease activity that double checks each nucleotide after it is added. This nuclease activity permits the enzyme to remove (hydrolyze) a newly added nucleotide and is highly specific for mismatched base pairs
- If the polymerase has added the wrong nucleotide, translocation of the enzyme to the position where the next nucleotide is to be added is inhibited
- This kinetic pause provides the opportunity for a correction, The 3' to 5' exonuclease activity cleaves the most recently added phosphodiester bond and removes the mispaired nucleotide. The polymerase then adds another nucleotide to begin synthesis again.
78
Compare DNA Pol 1 to DNA Pol 3:
1) 3' to 5' exonuclease activity (proofreading)
They both have this
79
Compare DNA Pol 1 to DNA Pol 3:
2) 5' to 3' exonuclease activity
Only DNA Pol 1
80
Compare DNA Pol 1 to DNA Pol 3:
Rates of polymerization
DNA Pol 3 (40) has a higher rate than DNA Pol 1 (10-20)
81
Compare DNA Pol 1 to DNA Pol 3:
Processivity
DNA Pol I has a lower processivity with (3-200) while DNA Pol II has a higher one with (1500)
82
What is the role of DNA Pol 1?
DNA Poly 1 is not the primary enzyme of replication, it performs a host of cleanup functions during replication, combination, and repair. Functions are enhanced by its 5' to 3' exonuclease activity
83
Why is DNA Pol III more processive than DNA Pol 1?
The Beta subunits acts like clamps and prevent the dissociation of DNA polymerase from the DNA strand, dramatically increasing processivity - to greater than 500,000
84
Helicase function
Enzyme that moves along the DNA and separates strands using chemical energy from ATP
85
Topoisomerase function
Relieves topological stress caused by strand separation
86
DNA binding proteins function
Separated strands are stabilized by DNA binding proteins (prevent denaturation)
87
Primase function
Enzyme that catalyzes the formation of RNA oligonucleotides used as primers
88
DNA Ligase
After an RNA primer is removed and the gap is filled with DNA, a nick remains in the DNA backbone in the form of a broken phosphodiester bond. These nicks are sealed by DNA Ligases
89
Discuss the sequence features at the origin of replication
- R and I sites: both bind DnaA. I sites can only bind DnaA when it has ATP
- IHF and FIS binding sites are for proteins
90
What is the function of the R and I sites? What happens at these sites that leads to unwinding of the double helix at the DUE?
- R and I sites serve as binding sites for the initiator protein, DnaA
- The tight right-handed wrapping of the DNA around the DnaA or R or I site creates a supercoil
******
91
What is the role of the helicase that enters?
Loading of the DnaB helicase is thet key step
- Migrates along the single stranded DNA in the 5' to 3' direction.
- DnaB helicases travel in opposite directions, creating 2 potential replication forks
92
As the fork enlarges, what enzyme continues to be involved in unwinding the double helix?
DnaB "Helicase"
93
Gyrase definition
A type II topoisomerase that introduces negative supercoils to counteract the positive supercoiling created outside of the replication bubble by the helicase
94
What protein bind to the resulting single stranded DNA? Why is this important?
SSB (single stranded binding proteins)
- Keeps single strand from renaturing to something else
95
What process is required before DNA synthesis can occur on the single-stranded template?
Primers must be on DNA template
96
What enzymes carry this out?
Primases
97
Why is RNA used as a primer instead of DNA?
RNA is used a s primer since it doesn't require a primer to be added to DNA
98
What enzyme now carries out the bulk of DNA replication?
DNA Polymerase
99
Discuss how a dimer of DNA polymerase at the replication fork can copy both parental strands simultaneously. Discuss the role of the beta subunit.
- Beta clamp is the purple ring and it keeps the polymerase from dissociating from teh DAN by forming a loop around it
- Helicase is moving along with the fork and primase will bind to the helicase and synthesize a little primer on the lagging strand. Then it will dissociated while our DNA polymerase will be finishing our other Okazaki strand
- Once the primer is completed, the clamp loading complex will load its next clamp onto the primer as DNA polymerase III is finishing the previous Okazaki fragment so it can attach to the new Beta-clamp and primer
- While DNA polymerase III is synthesizing this next strand, the Beta-clamp loading complex is preparing its next clamp to go on the next primer that is created
100
Name the DNA polymerase that removes teh RNA primer
DNA Polymerase I
101
Which activity from table 25-1 is used for RNA primer removal (draw an example to explain why this makes sense)?
5' to 3' exonuclease activity
This is because it's cutting from the 5' end. (in the 3' to 5' example, it was cutting from the 3' end)
102
Discuss the nick translation reactions used to remove the RNA primers
DNA ligase catalyzes the formation of a phosphodiester bond between a 3' -OH end at one of the DNA strands and the 5' phosphate at the end of the other. The DNA ligase must be activated by adenylylation. They use ATP in order to do this if they are DNA ligases from viruses or eukaryotes. Use NAD+ if its from bacteria
103
How is the DNA ligase activated?
By adenylylation
104
Name the enzyme that then catalyzes the formation of phosphodiester bonds between newly synthesized DNA fragments. To form the phosphodiester bond requires energy. Name the source of the energy
DNA ligase
- Viruses and eukaryotes use ATP
- Bacteria use NAD+
105
Draw out how the enzyme is adenylated and how it makes the phosphodiester bond
106
DNA replication is bidirectional. Use the diagram below to explain bi-directional DNA replication from a single origin of replication.
- The Ter sequences are arranged on the chromosome to create a blockade for the replication fork
- The Ter sequences function as binding sites for the protein Tus. The Tus-Ter complex squeezes together the DNA strands - thus preventing replication when the DNA polymerase gets to this part.
- Once the other replication fork meets the first one that was stopped, it will also be arrested
The point of this Ter sequence is so the DNA polymerase doesn't keep synthesizing parts of the DNA over again because it will continue to go in the circle.
107
What type of topoisomerase separates the catenanes?
Topoisomerase 4
108
Compare the number of origins of replication in E.coli to the number in eukaryotes
- E. coli only has one origin of replication
- Eukaryotes have more than one origin of replication
109
What are telomeres
The structures at the end of eukaryotic LINEAR chromosomes that consist of many tandem copies of a short oligonucleotide sequence. (repetition of TG nucleotides)
110
Discuss why linear chromosomes cannot be entirely copied by lagging strand synthesis
The lagging strand cannot complete replication at the very end of the chromosome because it needs to lay down a primer to make Ozakazi fragments, but there is no room to add the next primer because there is no template in that region
111
Name the enzyme that adds telomeric ends. What type of enzyme is it
Telomerase. It is a reverse transcriptase
112
What serves as the primer for telomere addition? What serves as the template?
- The TG (DNA) serves as the primer for addition (the 3' end)
- The RNA is the template
113
In humans, what types of cells (germline or somatic) have telomerase?
Germline cells
114
List three key processes that contribute to the high fidelity of DNA replication
1. Base selection by the polymerase
2. A 3'-5' proofreading exonuclease activity that is apart of many DNA polymerases
3. Specific repair systems for mismatches left behind after replication
115
Mismatch repair
Used to correct errors made by DNA polymerases that are not corrected by 3' to 5' proofreading exonucleases
116
Discuss how methylation is used to distinguish the parental strand in DNA replication from the newly synthesized strand
A bit after DNA is synthesized, the two strands get methylated so that when they are separated for replication, your enzymes know which strands are the parent and new strand
117
Discuss how mismatch repair works. How cells distinguish which nucleotide is the correct one when a mismatch is detected
- First, after some time of replication, the final "bow" at the top is to add methyl groups to both strands. This is how you can identify the parent strands
- Then MutS and MutL will form a complex and scan the DNA until they find a mismatch
- Once it finds the mismatch, the MutH will attach to the complex and find the nearest GATC site. The GATC should have a methyl on it, so it will notify the MutH which strand is the parent and new strand
- MutSLH will make a cut on the unmethylated strand to take out a chunk of DNA (including the mismatched region)
- DNA polymerase III will come in and repair the strand with all the things that it uses and DNA ligase will get rid of the nick
118
Base excision Repair: discuss the spontaneous cytosine deamination reaction
- Under typical cellular conditions, cytosine will become uracil spontaneously
- Bases may spontaneously lose amino groups
119
Base-excision repair: discuss base-excision repair naming each enzyme and discussing the function of each
- DNA glycosylase breaks N-Beta-Glycosyl bond
- AP endonuclease breaks the DNA backbone next to the empty spot
- DNA Polymerase 1 removes the bases from the 5' side and replaces them with new DNA bases leaving a nick that DNA ligase fixes
