Exam I Flashcards
1
Q
What are the components of a nucleotide?
A
Base, phosphate, sugar
2
Q
Nucleic acids contain what kind of sugars?
A
Pentose
3
Q
What is the difference between ribose and deoxyribose?
A
Ribose is found in RNA and has two -OH groups attached to the 2’ and 3’ carbons.
Deoxyribose is found in DNA and has one -OH group attached to the 3’ carbdon.
4
Q
What is the difference between a purine and pyrimidine? Give examples of bases.
A
Purines consist of two rings (Ex. Adenine and guanine)
Pyrimidines consist of one ring (Ex. Cytosine, thymine, uracil)
5
Q
What’s the difference between thymine and uracil?
A
A methyl group
6
Q
What is the name of the bond that connects the sugar to the base?
A
N-glycosidic bond (Bond between C and N, loss of water)
7
Q
What is the name of the bond that connects the sugar to the phosphate?
A
Phosphodiester bond (Bond between the “phosphonyl” P from the phosphate and the O from the sugar)
8
Q
What is the energy of a hydrogen bond?
A
~1 kcal/mol
9
Q
Where are the hydrogen bonds located in DNA?
A
Between the bases (A to T, C to G)
10
Q
What base pair is stronger and why?
A
G and C pairs are stronger because they have three H-bonds. A to T only have two H-bonds.
11
Q
DNA normally exists in __ form.
A
B
12
Q
DNA is a ___-handed helix.
A
right
13
Q
What are some characteristics of right-handed helixes?
A
~10 bp/turn, 3.4 nm per turn, 2 nm wide
14
Q
Why do minor and major grooves in DNA exist?
A
The strands run antiparallel to each other, so the N-glycosidic bonds are not equally opposite.
15
Q
Why are the phosphates in the phophodiester bonds of the two chains in DNA not diametrically opposite?
A
To minimize repulsion between negative charges
16
Q
Nucleic acid information can be “read” by what?
A
DNA binding proteins
17
Q
The alpha helix of a HTH (helix-turn-helix) domain can fit where in DNA?
A
The major groove
18
Q
The alpha helix of a ZFP (zinc finger protein) can fit inside where in DNA?
A
The major groove
19
Q
Beta sheets can fit inside where in DNA?
A
The major groove
20
Q
The amino acid side chains of DNA binding proteins can form _____ with the bases in the double helix.
A
H-bonds
21
Q
What is multivalency?
A
Multiple contacts increasing binding strength
(More H-bonds = Overall interaction is strong)
22
Q
If you stretched it out, how long would all the DNA in a human cell be?
A
1 meter
23
Q
Why do DNA dyes have a positive charge?
A
Nucleic acids have a negative charge.
24
Q
The accuracy of hybridization depends on what? Explain.
A
The temperature of annealing
Only stable hybrids form at higher temperatures (No mismatches).
Imperfect base pairing happens at lower temperatures (Has mismatches)
25
Explain FISH.
FISH stands for fluorescence in situ hybridization.
The FISH "probe" is a complementary sequence + fluorescent dye (FL).
FISH applications include detection of translocations in cancer and analysis of gene localization.
26
Explain gel electrophoresis.
DNA is chopped up into smaller pieces using a restriction enzyme.
DNAs of different lengths migrate proportionally to their length. The shortest DNAs move fastest.
DNA is stained with intercalating dye to give a brighter signal.
27
Explain Southern & Nothern blots.
Watch a video.
28
What is recombinant DNA?
Artificially created DNA that combines sequences that do not occur together in nature
29
Why clone DNA?
To identity genes, express genes, mutate genes, and sequence genomes
30
What is the difference between endonucleases and exonucleases?
Endonucleases hydrolyze DNA in the interior of double helices.
Exonucleases hydrolyze DNA from the ends.
31
Restriction enzymes are what kind of nuclease enzyme? What do they do?
They're deoxyriboendonucleases that recognize specific DNA sequences and hydrolyze a phosphodiester bond at specific sequences.
32
Many restriction enzymes recognize DNA palindromes. What are palindromes?
DNA palindromes read the same forward and backward.
33
Restriction enzymes leave what kind of ends? What does this allow for?
They leave overhanging, sticky ends, allowing for more specificity because they can base pair.
34
What is the function of ligase?
Covalently joins two DNA fragments (Usually in DNA repair)
35
Where do restriction enzymes come from?
They're made by bacteria to defend themselves from foreign DNA.
Bacteria protect their DNA by methylating the related recognition site to prevent their hydrolysis.
36
What's a polylinker?
Multiple cloning site; a DNA segment with several unique sites for restriction enyzmes
37
What are genomic libraries?
Collection of chimeric plasmids that include all DNA in a genome
38
What is the function of reverse transcriptase?
It's a DNA polymerase that uses RNA as a template.
39
Eukaryotic mRNAs end with a string of what?
A bases (Poly A tail)
40
By what method is an RNA:DNA hybrid converted to DNA:DNA using DNA polymerase?
Polymerase chain reaction (PCR)
41
Learn the steps of PCR.
Ok
42
What is the function of DNA polymerase?
Catalyzes the addition of a nucleotide unit using the principle of base complementarity
43
What is a primer?
It requires a chain that base pairs with the template and has a 3' -OH on which to add
44
In Sanger sequencing, how does DNA replication stop?
DNA replication stops without a 3' -OH on the sugar. This prevents strand extension.
45
What is a CONTIG?
A series of overlapping DNA sequences used to make a physical map that reconstructs the original DNA sequence.
Software algorithms can recognize the overlap between clones. This builds a CONTIG.
46
What are the three components useful for cloning vectors?
1. Replication origin
2. Selectable markers (Drug resistance, nutritional marker)
3. Cloning site (Restriction enzyme recognition site)
47
What is the start codon?
AUG
48
What are some stop codons?
UAA, UAG, UGA
49
What is the coding strand?
The DNA strand that has codons in their DNA form
50
What is the template strand?
The strand read by RNA polymerase to make a complementary RNA strand
51
Only __% of our genome encodes for proteins.
1
52
25% of our genome consists of what?
Exons and introns
53
The remaining 75% of our genome is what?
Spacer DNA or intergenic DNA
DNA between genes that can be unique or repetitive (Outside of DNA sequence of a gene)
54
What is the function of the TATA box?
TATA box binding proteins recognize TATA sequence and promotes transcription.
55
What are DNA-only transposons?
Cut-and-paste gene from one area of genome to another (Fossilized in our genome and no longer active)
56
What are the two types of retrotransposons?
Retroviral-like: Copy-and-paste genes also fossilized in our genome
Nonretroviral: Copy-and-paste genes; some sequences are still active and jump around the genome
57
What are retrotransposons derived from?
Viral genetic material integrating into our own genome
58
Retrotransposons make up about __% of our genome.
40
59
Nonretroviral retrotransposons are divided into what two groups? Explain.
LINEs: Long interspersed nuclear elements
Sequence encodes its own RNA binding protein and reverse transcriptase/endonuclease
SINEs: Short interspersed nuclear elements
Does not code for proteins and relies on L1's reverse transcriptase
60
What is the only LINE left in the human genome?
L1 element
61
What is the most common SINE?
Alu
62
What are pseudogenes?
Gene copies that have mutations that inactivate gene expression
63
Where does replication begin?
Origin
64
What are the substrates and products of a reaction with DNA polymerase?
Substrates: Template strand and dNTP
Products: Nascent strand and pyrophosphate
65
Before DNA polymerase can bind DNA, it needs a ______.
primer
66
How does DNA polymerase work?
It catalyzes the formation of phosphodiester bonds between the dNTP and growing strand.
67
What are the four DNA polymerases in prokaryotes?
DNA polymerase I
DNA polymerase II
DNA polymerase III
Translesion polymerases
68
What are the six DNA polymerases in eukaryotes?
DNA polymerase alpha
DNA polymerase beta
DNA polymerase gamma
DNA polymerase delta
DNA polymerase epilson
Translesion polymerases
69
What is the function of DNA polymerase I?
DNA polymerase I: Essential; used in Okazaki fragment joining
70
What is the function of DNA polymerase II?
DNA polymerase II: Used mainly in DNA repair
71
What is the function of DNA polymerase III?
DNA polymerase III: Used for both leading and lagging strand replication
72
What is the function of translesion polymerases?
Translesion polymerases: Used to polymerize over damaged bases
73
What is the function of sliding clamps?
The clamp binds to DNA polymerase and makes it processive, keeping it bound to DNA.
74
Sliding clamps are loaded by what?
Clamp loaders
75
What is the function of primase?
It's a type of RNA polymerase that creates an RNA primer on DNA, so DNA polymerase can bind.
76
What is the function of DNA ligase?
DNA ligase joins the Okazaki fragments.
77
What is the function of DNA helicase?
DNA helicase unwinds the DNA by circlng one of the strands and traveling along that to unwind the helix and force apart the H bonds.
78
What is the function of single-strand binding protein?
It prevents strands from rewinding and folding up into multiple hairpin loops.
79
What is the function of topoisomerase?
It relieves supercoiling by allowing the DNA to rotate and relieve accumulated strain.
80
How does DNA polymerase make only a few mistakes?
Most DNA polymerases have a 3'-5' proofreading exonuclease that removes mismatched bases.
81
How is DNA replication initiated in bacteria?
Mutiple copies of membrane associated initator protein (DnaA) is bound to the DNA.
82
What controls origin firing in prokaryotes?
Methylation: Hemimethylated (one strand) origins are resistant to initiation. Only fully methylated origins are competent for initiation.
83
What does the protein Tus do?
Tus binds to the Ter sequences and acts as a funnel, allowing the replication fork to only go forward.
84
Summarize the initation of DNA replication in prokaryotes.
1. Origin is methylated.
2. Initiator proteins bind.
3. AT-rich region structurally opens up.
4. Helicases are loaded to AT-rich region and activated.
5. DNA primase is loaded and activated.
6. RNA primers enable DNA polymerase activation.
85
Summarize the termination of DNA replication in prokaryotes.
1. Tus proteins let the replication complex pass one-way only.
2. Both replication complexes reach the middle and can't pass each other.
86
What is the difference between eukaryotic and prokaryotic DNA in regards to replication origins?
Eukaryotic chromosomes are linear with multiple replication origins.
Prokaryotic DNA is circular with one origin.
87
What are the two components of yeast replication origins?
1. A sequence where a protein complex (ORC) binds
2. An AT rich region that can be easily unwound
88
DNA is replicated once during the __ phase of the cell cycle.
S
89
The ORC complex is _____ and binds to thousands of locations along our chromosomes.
conserved
90
Name two ways that re-replication is prevented.
1. Origins are first "licensed" by the assembly of a pre-replication complex (pre-RC) under conditions that prevent initiation.
2. A subset of the pre-RCs are activated (origin activation) under conditions that prevent pre-RC formation.
91
Name the five steps to origin licensing.
1. ORC (origin recognition complex) binds to the origin.
2. CDC6 and CDT1 load the helicase complex constituted by a double hexamer of MCM2-MCM7.
3. The hexameric Mcm helicase is loaded by the loaders Cdc6 (binds to ORC) and Cdt1 (binds to MCM) to form the pre-replication complex (pre-RC).
4. Pre-RC is inactive and wrapped around double-stranded DNA in an stable complex.
5. Cdc6 is phosphorylated and then degraded by the proteasome.
92
What happens during origin activation?
Activation of the helicase complex requires phosphorylation of the MCM by Dbf4-dependent kinase (DDK)
93
What happens during origin firing?
Once the MCM helicase complex is phosphorylated it “fires” splitting the complex into two sub complexes that move away from each other establishing the replication forks.
94
What is replication timing?
The temporal order in which distinct segments of the genome are replicated.
95
What are the ends of our chromosomes called?
Telomeres
96
What is the function of ribonucleoprotein?
It brings its own RNA to use as a template.
97
What is the function of telomerase?
It adds telomeric repeats (TTGGGG) to maintain telomere length.
98
In humans, the mutation of telomere maintenance genes results in what?
Dyskeratosis congenita (DC)
Correlates with extreme telomere shortening
99
Cells do what when their telomeres become too short?
They senesce ("dye")
100
What is the Hayflick limit?
The number of times a normal cell can divide
101
Only __% of our DNA comprise codons.
1.5
102
Two copies of each of your DNA molecules are not the exactly same (98-99% the same). Most of these differences are inconseqential and are called what?
Polymorphism
103
Most polymorphisms are what?
Single base pair differences called SNP (single nucleotide polymorphism)
104
What is an example of transition and transversion?
Transition: A:T to G:C (Purine for purine)
Transversion: A:T to T:A (Purine for pyrimidine)
105
What are CNVs or copy number variations?
Segmental duplication; variations of kb length segments in chromosomes
106
What is a translocation?
The moving/exchanging of chromosome sections
107
What's a missense mutation?
The substitution of a codon for another that codes a different amino acid
108
What's a nonsense mutation?
Substitution of a codon encoding for an amino acid with a stop codon
109
What's a frameshift mutation?
Insertion or deletion of a nucleotide in which the number of deleted base pairs is not divisible by three
110
Causes of mutations?
1. Chemical environment of cells (Oxygen/oxidation)
2. Environmental insults (UV light, x-rays)
3. Enzymatic mistakes
111
What are the repair mechanisms for lesions affecting a single strand?
1. Mismatch repair (MMR)
2. Base excision repair (BER)
3. Nucleotide excision repair (NER)
4. Translesion polymerases
112
What are the repair mechanisms for lesions affecting both strands (double strand breaks)?
1. Homologous recombination (HR)
2. Non-homologous end joining (NHEJ)
113
What happens when there is U in DNA?
Uracil DNA glycosylase detects and removes it when present.
114
What is the mechanism of BER?
Poorly matched bases are recognized and rotated out of the helix, recognized by glycosylase.
115
How do translesion polymerases work?
The sliding clamp releases DNA polyemerase, and translesion DNA polymerase is loaded and bypasses the DNA damage, continuing synthesis.
116
What are the three functions of homologous recombination?
1. Assists in DNA repair
2. Links sister chromosomes to properly segregate them between self and daughter cells
3. Source of DNA exchange and genetic diversity
117
What do RecA and Rad51 do?
They use the 3'-end of the broken DNA to invade the homologous duplex in HR. They can take a broken DNA end and search for a homologus duplex.
