Exam 1 Flashcards
(136 cards)
1
Q
What is a nucleoside?
A
A base and a sugar – NO phosphate
2
Q
What is a nucleotide?
A
A base, a sugar, and a phosphate group
3
Q
What is the name of the bond which attaches two nucleotides together?
A
Phosphodiester bond
–> Forms between the 5’ end of one sugar and the 3’ end of the next sugar
4
Q
What is the name of the bond which holds together two phosphate groups?
A
Phosphoanhydride bonds
5
Q
True or false: Phosphoanhydride bonds are not easily hydrolyzed
A
FALSE: These bonds ARE easily hydrolyzed. When the bond is hydrolyzed, energy is released
6
Q
What three stabilizing forces are present in B DNA?
A
i) Placement on opposite sides of the double helix (on outside of helix, as opposed to inside)
ii) The negative charge on the phosphates are neutralized by positive charges of lysine and arginine residues of histone proteins
iii) The stacking of the aromatic rings of the bases that permits van der Waal interactions between the electron clouds that sandwich the rings
7
Q
What is the bond between the base and the sugar in a nucleoside?
A
N-glycosidic bond
8
Q
How does A form differ from B form? What type of DNA or RNA takes on the A form?
A
- Wider and more compact than B
- The structure assumed by dsRNA (double-stranded RNA)
9
Q
How does the Z form differ from the B form? What type of DNA or RNA takes on the Z form?
A
- It is left-handed as opposed to right-handed
- Doesn’t really exist in nature
10
Q
Describe 3-stranded DNA
A
-Occurs through Hoogsteen base pairing (H-binding with N’s in 5-membered (rather than 6-membered) ring from purine)
11
Q
What is 4-stranded DNA? Where does it occur?
A
- Called G quartets or G-quadruplexes
- Occurs in G-rich areas of DNA or RNA
- Stack on top of each other in 4G coils
- Occurs often in telomeres
12
Q
What is ssDNA? Where would you find it?
A
- It is circular DNA (bound by a phosphodiester bond from the 3’ to 5’ end)– Also called a plasmid
- Found in bacteria and archaea
13
Q
Which part of a helix-turn-helix motif can fit into the major groove of DNA?
A
The a-helix
14
Q
What part of a zinc finger motif can fit into the major groove of DNA?
A
The a-helix
15
Q
How do DNA binding proteins work?
A
They recognize a specific sequence of DNA and can bind in the major groove
16
Q
True or false: B-sheets can fit into the major groove of DNA
A
TRUE - B-sheets, along with a-helices, can both fit into the major helix of DNA in order to interact with it
17
Q
How does a protein attach to DNA when interacting with it?
A
Through hydrogen bonds on the amino acid side chains of the protein and the phosphate backbone of the DNA (in the major groove)
18
Q
Definition: Multivalency
A
When a lot of weak bonds all work together and the resulting bond of the two molecules is very strong (think of velcro– only one fiber of velcro is weak, but the more you have, the better the two objects stick together)
19
Q
What is the molecular basis by which proteins read DNA’s encoded information? (3 answers)
A
i) Hydrogen Bonding
ii) Multivalent interactions
iii) Intercalation
20
Q
What does a restriction enzyme do?
A
Restriction enzymes chop up DNA
21
Q
What does an intercalating dye do? What is an example of an intercalating dye?
A
- And intercalating dye stains DNA so that it can be seen under UV light (typically in a gel electrophoresis experiment)
- Ethidium bromide is an intercalating dye
22
Q
What is hybridization?
A
When a set of bases pairs with a complementary set of bases (making hydrogen bonds)
–> When one strand of DNA binds to a new strand of DNA (not when a new strand is being formed from a template strand)
23
Q
Why do higher temperatures create the more accurate hybridizations?
A
Only perfect matches will form at higher temps because they are the only ones strong enough to resist the heat– weaker (imperfect) matches will be unable to stay bonded at high temps
24
Q
What is a Western Blot used for?
A
To detect proteins
25
What is a Northern Blot used for?
To detect RNA
26
What is a Southern Blot used for?
To detect DNA
27
Definition: DNA sequencing
The technology to determine the order of base pairs along DNA molecules
--> Often requires cloning
28
When cloning via plasmids, why must you use the same restriction enzymes for both the plasmid and the DNA?
You need to use the same restriction enzymes for both so that they have matching sticky ends. The sticky ends will improve binding.
29
What are the two types of "Libraries"? What is the difference between them?
i) Genome Library
- -Contains all genes in a genome, both coding and non-coding
ii) cDNA Library
- -Contains ONLY genes that code for proteins (exons)-- no introns or non-coding areas are included
30
Describe blue-white colony selection using the LacZ gene
- Plasmids are made that should disrupt the LacZ gene when inserted into the bacterial genome
- After mixing plasmids and bacteria, the bacteria are grown on a plate that contains a blue galactosidase compound
- Those bacteria that grow into a blue colony can still process galactosidase (because their LacZ gene is still intact), and therefore these are UNDESIRABLE colonies (do not contain the desirable plasmid)
- Colonies that grow as a white color have a disrupted LacZ gene, therefore these colonies are selected FOR, because they have the desirable plasmid
31
What is an oligo dT? What is its purpose?
Basically a 'poly-T' situation-- bunch of Ts in a row (can bond to a poly-A tail)
--> Used as a universal primer for cDNA cloning
32
True or false: You can create tissue-specific cDNA libraries
TRUE: Different genes are expressed in different tissues, and in creating a cDNA library, only the genes that are expressed in a given cell are reverse transcribed into cDNA
33
What is a restriction enzyme? What do scientists use them for?
- Restriction enzymes are used by other organisms to degrade foreign DNA (a type of immune system)
- We (scientists) use them to break up long DNA molecules into manageable-sized pieces
34
What does an endonuclease do? What does an exonuclease do?
Endonucleases hydrolyze DNA in the interior of a double helix
-Exonucleases hydrolyze DNA from its ends
35
What type of endonucleases do we use to break apart DNA molecules?
Deoxyriboendonucleases
36
True or false: You can use PCR on DNA and proteins
FALSE: You can ONLY use PCR for DNA (also RNA). Proteins cannot be PCR'ed because during the heating step of PCR, the proteins will denature
37
How do you selectively amplify a gene in PCR?
By adding in primers for those specific sections of DNA or RNA
38
What component properties are needed in a vector?
i) Selection Marker Genes (like the lacZ gene or a drug resistant gene)
ii) Origin of Replication (NOT all vectors require a promoter, but a typical vector does require one)
iii) Restriction Site (place where the genome gets chopped in order to insert the new stuff)
39
What does BAC stand for?
Bacterial Artificial Chromosomes
40
What is an MCS? What does it stand for?
- Stands for Multiple Cloning Site
| - An artificial sequence that contains the recognition sequence for different restriction enzymes
41
Explain Dideoxy Sequencing (also called Sanger Sequencing)
Shortest strands go through first, we can see what color is at the truncated end. So it would be like 1 nucleotide long, 2 nucleotide long, 3 nucleotide long, etc. and based on what colors come through one after the other you can see what the sequence is
42
What is a CONTIG?
A contiguous overlapping sequence
--Assembling a bunch of CONTIGs is what you must do if you don't know anything about your sequence
43
Describe Illumina Sequencing
* Break up DNA into small pieces (~100 bp)
* Attach adapters to both ends
* Denature and attach both ends to an optical plate
* Make DNA double-stranded with DNA polymerase
* Denature to make single-stranded
* Amplify
* Carry out DNA sequencing with primers, DNA polymerase and fluorescent nucleotides
* Reads often do not go for more than 25 nucleotides
44
What is SKY?
Getting an entire chromosome labeled with a specific color (each chromosome having a different color to easily differentiate)
- -Whole genome FISH
- -Can reveal genetic abnormalities
45
Approximately what percentage of the human genome codes for proteins?
1.5%
--About 25% of made up of genes, but most of this is introns which get removed before translation
46
What is the start codon (starts TRANSLATION)?
AUG -- Codes for methionine
--> This is also the only methionine codon, so it gets used not only as the initiator codon but also to code all methionines in proteins
47
What are the three stop codons (terminates TRANSLATION)?
UAA, UAG, UGA
--Do not code for anything else except stopping translation
48
When do introns get removed from the genome?
Introns are removed as pre-mRNA is processed to mRNA
49
What is spacer DNA?
DNA between genes that can be unique or repetitive
50
True or False: DNA Transposons are the primary transposon type in humans
FALSE: DNA transposons are called 'fossilized' transposons because they exist in our genome, but are no longer functional
The primary type of transposon in human genomes is nonretroviral transposons
51
How do nonretroviral transposons increase the length of the genome?
The receiving end has little overhangs that the transposon binds to. These overhangs get duplicated when the transposon is inserted, and this lengthens the genome
52
True or False: Retrotransposons use RNA to move genetic material around
TRUE - Use of reverse transcriptase
53
What does LINE stand for? SINE?
LINE: long interspersed nuclear elements
SINE: short interspersed nuclear elements
54
Aside from length, what is the major difference between LINEs and SINEs?
LINEs are autonomous, SINEs are not
--> Autonomous means that the sequence encodes its own RNA binding protein and reverse transcriptase (called L1 in humans-- SINEs don't make their own, depend upon LINEs creating enough L1 for both)
55
How does unequal crossover occur?
Because of accidental "slippage"/misalignment during recombination (during e.g. meiosis)
56
What is synteny?
Same relative order of genes in different species -- shows evolution occurs
57
What does an enzyme do?
Orientates two substrates in the right manner in order to push forward a chemical reaction
58
What does DNA polymerase do?
Catalyzes the formation of phosphodiester bonds between the dNTP and the ‘growing strand’
--> Adds nucleotides to the growing strand of DNA
59
DNA polymerase requires energy in order to function. Where does the energy come from?
This enzymatic reaction uses energy from the ‘high-energy’ phosphoanhydride bonds from the dNTP (breaks the dNTP to dNDP)
60
True or false: DNA polymerase is an enzyme
TRUE
61
What are the substrates in the reaction that DNA polymerase catalyzes? What are the products?
Substrates: Growing strand and dNTP
Products: Growing strand covalently bonded to dNMP and pyrophosphate
62
In what direction does DNA polymerase add new nucleotides to the growing strand?
5' --> 3' ALWAYS
63
What does dNTP stand for?
Deoxynucleotide Triphosphate
64
What does DNA polymerase I do?
Used in Okazaki fragment joining (removes RNA primer and replaces it with DNA)
-ONLY in prokaryotes
65
What does DNA polymerase II do?
Used mainly in DNA repair
| -ONLY in prokaryotes
66
What does DNA polymerase III do?
Used for both leading and lagging strand synthesis
| -ONLY in prokaryotes
67
What do translesion polymerases do?
Used to polymerize over damaged bases
| -Both prokaryotes and eukaryotes
68
What does DNA polymerase alpha (a) do?
Okazaki fragment initiation
| -ONLY in eukaryotes
69
What does DNA polymerase beta (B) do?
Used mainly in DNA repair
| -ONLY in eukaryotes
70
What does DNA polymerase gamma do?
Mitochondrial DNA replication
| -ONLY in eukaryotes (duh-- no mitochondria in prokaryotes)
71
What does DNA polymerase delta do?
Lagging strand replication (NOT leading strand!!)
| -ONLY in eukaryotes
72
What does DNA polymerase epsilon do?
Leading strand replication (NOT lagging strand!!)
| -ONLY in eukaryotes
73
What does primase do?
A type of RNA polymerase which creates an RNA primer on DNA; then DNA polymerase can bind.
RNA primers are 10 nucleotides long
74
Why are RNA primers used instead of DNA primers in DNA replication?
- Primase is one of the most error prone and slow polymerases
- RNA primers can be recognized later and replaced with DNA by a less error prone polymerase
75
What does DNA ligase do?
Makes an intermediate at the 5’ phosphoryl of the nick. The 3’-OH at the nick then carries out a nucleophilic attack to join the 3’- and 5’- hydroxyls of the DNA in a phosphodiester bond
76
What does DNA helicase do?
- Hydrolyzes ATP to ADP + Pi
| - They circle one of the two strands and travel along that strand unwinding the helix by forcing apart the H bonds.
77
What do single stranded binding proteins do?
- Prevents strands from re-annealing in DNA before replication
- Coats the single strands behind helicase unwinding to prevent immediate re-bonding to the complementary DNA strand
78
What do topoisomerases do?
Relieves supercoiling in DNA replication
79
What is the proofreading exonuclease?
- Part of most DNA polymerases
| - Performs proofreading of newly synthesized strand in a 3'--> 5' direction
80
What does DnaA do?
- Hydrolyzes ATP (in E. coli)
- DnaA is a protein that activates initiation of DNA replication in bacteria. It is a replication initiation factor which promotes the unwinding of DNA at oriC. The onset of the initiation phase of DNA replication is determined by the concentration of DnaA
81
How does E. coli prevent replication from starting multiple times before the whole chromosome is duplicated once?
Methylation of the parent strand, while preventing methylation of the daughter strand until the entire chromosome is duplicated
--> There is a GATC in a 13-bp repeat, and it is the A that is methylated, not every single nucleotide
82
What does the inhibitor protein in E. coli replication do?
It is an inhibitor protein on the hemimethylated origins. Helps to make sure that the refractory phase stays long enough (if it doesn't stay long enough, the cell will begin replicating DNA again before it is finished).
83
How does E. coli stop replication when the two forks meet?
A protein named Tus binds to the Ter sequences and acts as a funnel allowing the replication fork to go “forward” but not “backwards”.
84
Give a summary of prokaryotic replication initiation
– Origin is methylated
– Initiator proteins bind
– AT-rich region structurally opens up
– Helicases loaded to AT-rich region and activated
– DNA primase loaded and activated
– RNA primers enable DNA polymerase activation and replication start
85
Give a summary of prokaryotic replication termination
– Tus proteins let replication complex pass one-way only
| – Both replication complexes reach the “middle” and can’t pass each other OR the opposite-facing Tus proteins
86
How does a micro-array work?
Immobilize known sequences on a chip & then DNA that's floating around can be hybridized to the chip to see what's in solution at any given time
87
During what phase of the eukaryotic cell cycle is DNA replicated?
S phase
88
What is the ORC?
- Origin recognition complex (binds to origin of replication)
- Consists of six subunits. It binds to the origin. It hydrolyzes ATP.
- Unwinds AT-rich region in order to allow Mcm helicase to bind
- In EUKARYOTES
89
What is the pre-RC?
- The hexameric Mcm helicase (phosphorylation for activation) is loaded by the loaders (next to the ORC)
- Cdc6 (ORC binder, (phosphosylated for degradation)) and Cdt1 (Mcm binder, phosphosylated for degradation) to form the PREReplication Complex (pre-RC)
90
Describe the final step of origin licensing in eukaryotes
- Cdc6 is phosphorylated and then degraded by the proteasome.
- ORC is also phosphorylated (causes it to fall off origin of replication, but then it will re-bind while still phosphorylated)
- The degradation of Cdc6 and the phosphorylation of ORC prevents origins from firing more than once.
91
During what phase of the eukaryotic cell cycle does the pre-RC form?
G1 phase
92
True or False: The helicase-loading proteins Cdc6 and Cdt1 are only active in S phase.
FALSE: Active in G1 phase
93
True or False: The helicase Mcm1 can be delivered to the origin recognition complex (ORC) only in S phase.
FALSE: Occurs in G1 phase
94
True or False: The ORC can only become active (by dephosphorylation) in G1 phase.
TRUE
95
True or False: The helicase can only become active (by phosphorylation) in S phase.
TRUE
96
True or False: The ORC can bind to the origin only in S phase.
FALSE: Binds in G1 phase, Then unbinds and binds again in S phase, and it is bound during the entirety of G2 phase
97
Which histone proteins stay associated during replication?
H3-H4 tetramers
– “Old” ones stay associated
– Some new are needed
98
Which histone proteins are completely released during replication?
H2A-H2B dimers
– Released completely
– A mixture of old and new is added back
99
How do proteins chaperones assist in replication of DNA?
Protein chaperone complex helps assemble histone proteins
– Chromatin assembly factors
– Bind to PCNA sliding clamp
100
What is replicative senescence?
When telomerase expression is lost and telomeres are gone & the actual genome gets chewed up
101
What is telomerase?
-It is a ribonucleoprotein: brings its own RNA to use as a template
– Adds telomeric repeats (TTGGGGTTGGGGTTGGGGTTG) to maintain telomere length
102
What are the two telomere capping mechanisms?
- G-quadruplexes and capping proteins
| - t-loops and capping proteins
103
What is a t-loop?
Strand invading & exchange to prevent a single-stranded end (on telomeres)
104
What is shelterin?
Shelterin covers the telomere to make sure that the single-stranded end is not recognized as a single-strand break & fixed improperly
105
What is a SNP?
- Differences of a single base pair difference from one DNA molecule to another
- Stands for single nucleotide polymorphism
- The term SNP is sometimes replaced by SNV for single nucleotide variation.
106
What is a transition?
A purine mutated to another purine OR
| A pyrimidine mutated to another pyrimidine
107
What is a transversion?
A pyrimidine mutated to a purine or vice-versa
108
What is an indel?
An insertion or a deletion
| --Includes one or multiple added or lost bps
109
What is a CNV?
- Stands for Copy Number Variation
- Also called segmental duplication
- Are variations of kb length segments in our chromosomes
110
What is a translocation?
A type of GCR (gross chromosomal rearrangement) in which sections of chromosomes are moved or exchanged
111
What four repair mechanisms only work on single-stranded DNA (nicks or gaps)?
– Mismatch Repair (MMR)
– Base Excision Repair (BER)
– Nucleotide Excision Repair (NER)
– Translesion polymerases
112
What two repair mechanisms work on double stranded DNA breaks?
- Homologous Recombination (HR)
| - Non-homologous End Joining (NHEJ)
113
What three subunits make up the mismatch repair mechanism?
MutS, MutL, MutH
114
What does MutL do? (Mlh in eukaryotes)
Associates with MutS and removes the damaged strand
115
What does MutS do? (Msh in eukaryotes)
Associates with MutL. MutS is what binds to the strand and scans for single-stranded nicks
-Creates a bubble in the DNA
116
What does MutH do (it only exists in prokaryotes)?
Recognizes hemimethylation. Creates a nick in the damaged strand of DNA for the MutS/MutL complex to recognize that there is an error
--> Is attached to MutL, but runs along in front of the MutL and MutS
117
In mismatch repair, after the strand of damaged DNA is removed, how is it replaced?
DNA polymerases replace the missing DNA and DNA ligase binds the new section into the double helix
118
What enzyme removes uracils in DNA?
Uracil DNA glycosylase
119
How does base excision repair work?
- A glycosylase recognizes the mismatched base and cuts out ONLY the base (backbone still there)
- The “bare” backbone is then cut out by AP (apurinic/apyrimidinic) endonuclease and Phosphodiesterase
- DNA polymerase replaces the base
120
What is the set of enzymes used in nucleotide excision repair in PROkaryotes?
Carried out by a set of enzymes and proteins encoded by the genes called UvrABCD
121
What is the set of enzymes used in nucleotide excision repair in EUkaryotes?
-Carried out by enzymes and proteins encoded by a set of genes named after a rare hereditary disease XPA-G (XPA, XPB... XPG)
-Excision cuts are 24-36 nt apart
36
122
Which repair mechanism uses DNA helicase to open up DNA to remove incorrect bps?
Nucleotide excision repair
| -This happens for both eukaryotes and prokaryotes
123
Describe nucleotide excision repair
- Fixes dimers (i.e. thymine dimers)
- An excision nuclease cleaves the strand of DNA containing the dimer, about 12 bps apart in eukaryotes, 24-36 bps apart in eularyotes
- DNA helicase comes by and removes the nicked-out bit of DNA
- DNA polymerase and DNA ligase come in and fill in the gap
124
What four things can cause a double-strand break?
- X-radiation (AKA ionizing radiation)
- breakage of single stranded templates during replication
- Endonucleolytic cleavage during meiosis
- Mitotic forces acting on telomere fusions
125
When can homologous recombination occur in the cell cycle?
Only when the DNA is replicating
-If the double strand break occurs during or after DNA replication, we can use the sister chromatid for sequence information
126
Describe how the strands of a double-break can find new template strands
- RecA (in prokaryotes) and Rad51 (in eukaryotes) use the 3'-end of the broken DNA to invade the homologous duplex (THIS REQUIRES ATP)
- The 3'-end serves as a primer for DNA polymerase. Missing pieces are built
127
True or False: Holliday junctions are covalently bound
TRUE: This makes it difficult to remove the invading DNA, so the invading DNA just combines with the template DNA and it is cut that way
128
What are potential issues with homologous recombination?
- When HR works well, it is very good at accurately repairing using the homologous copy it can use as a template
- When it doesn't work (can't find an accurate template), translocations, deletions, and inversions can occur
- Loss of heterozygosity is another potential issue
129
What mechanism is used to paste transposons into the genome?
Non-homologous End Joining
130
What protein acts as a marker on DNA to notify that there is a double-strand break?
A phosphorylated histone called H2AX
| --> These histones accumulate at the site of damage
131
What can be used to induce de-differentiation of stem cells?
Specific transcription factors
| KLF4, OCT4, SOX2 & C-MYC
132
What is the protein that cuts DNA?
Nucleases (endonucleases, exonucleases, etc)
133
True or False: Zinc Fingers contain both the DNA recognition and cleavage functionalities
FALSE: MEGANUCLEASES contain both the DNA recognition and cleavage functionalities.
Zinc fingers (and all other nucleases) only contain the DNA recognition function
134
To what endonuclease are Zinc Fingers and other nucleases associated in order to cut the DNA?
Fok1 endonuclease cuts the DNA; The zinc finger recognizes the DNA that needs to be cut
135
What is the name of the endonuclease that cleaves in CRISPR?
Cas9 (CRISPR is the DNA recognition part)
136
How does CRISPR recognize the sequence that it should cleave?
Guide RNA
