1
Q
What are the phases of the eukaryotic cell cycle?
A
Go, G, S, G2, and M
2
Q
What is the Go/G1 phase?
A
Cells do not actively divide and it is the first growth phase of the cell
3
Q
What is the S phase?
A
cell replicates entire genome
4
Q
What is the G2 phase
A
second growth phase before division
5
Q
What is the M phase?
A
cell divides into two identical daughter celsl
6
Q
Who was Arthur kornberg?
A
a biochemist who made several crucial discoveries with respect to eh proteins that generate the DNA polymer, along with how this process occurs
7
Q
What were some of Kornberg’s discoveries?
A
developed an assay for measuring the amount of DNA synthesis that is occurring using bacteria cell extracts
discovered several heat stable factors are needed for synthesis which are nucleotide triphosphates
purified DNA polymerase using multistep fractionation
8
Q
What was Arthur Kornberg’s work described as?
A
The beginnings of biotechnology
9
Q
What was Kornberg’s purification of the polymerase?
A
a multistep process where protein concentration and specific enzyme activity were monitered. Purified from bacterial lysate. Proteins were monitored in the fractions for the ability to perform a certain function
10
Q
What were the steps of purification of the polymerase?
A
I: break bacteria by signification
II-V: removed DNA and RNA
VI: used ammonium sulphate to precipitate some proteins and leave others in solution based on protein properties
VII: ion exchange chromatography to give a more purified product
11
Q
What was done at each stage of the experiment t purification?
A
measurement of protein content in mg/ml via spectroscopy, SDS PAGE and additional assays. specific activity of the fraction was measured. Activity increases the purer the protein
12
Q
What was the requirements of polymerization experiment?
A
determined components critical for DNA replication by determining the maximal incorporation of deoxyribonucleotides into DNA under various conditions.
13
Q
How was the requirements of polymerization experiment accomplished?
A
by performing 8 experiments. Each one, they removed one or more factors and used radio labeled dTTP to monitor the effects of deoxyribonucleotide incorporation into the DNA
14
Q
What were the requirements of polymerization results?
A
every experiment where a factor was removed resulted in an almost complete loss of incorporation.
All 4 nucleotides must be present
DNA template must be intavt
Mg++ cofactor for teh polymerase
15
Q
What was the key replication component Kornberg purified?
A
DNA polymerase I
16
Q
What are the six general principles of DNA replication?
A
DNA replication is semiconservative
replication is initiated at specific sites
replication is typically bidirectional
replication is semidiscontinuous
RNA primers are needed to start replication
Nuclease, polymerase’s and ligases replace the RNA primers with DNA and seal the nick
17
Q
What is conservative replication?
A
parental strands remain together and daughter is completely new
18
Q
What is diseprsive replication?
A
the parental strand and the new daughter strand will be randomly mixed together creating new strands having a patchwork of old and new DNA
19
Q
What is semiconservative replication?
A
Watson and crick hypothesized that helix must be unwound for replication and each molecule gets one parent and one daughter strand
20
Q
What is the origin of replication?
A
The specific site at which DNA synthesis begins with the participation of origin replication system
21
Q
What is the replication fork?
A
the point where the parental duplex seaprates
22
Q
What is the replication bubble?
A
The open DNA
23
Q
What does bidirectional replication fork mean?
A
The fork moves away from the origin in both directions
24
Q
What direction does DNA pol I synthesize>
A
5’ to 3’
25
What is the leading strand?
Synthesized continuously along the 3'-5' DNA
26
What is the lagging strand?
discontinuous synthesis along the 5' to 3' strand
27
What are the fragments from the lagging strand called?
Okazaki fragmenta
28
What is semidiscontinuous replication?
one lagging and one leading strand
29
How is DNA linked?
by the phohsphodiester bond by linking the a-5'-phosphate of the new nucleotide and the 3' OH.
30
What are primases?
enzymes that synthesize primers where they are required
31
What re nucleases?
enzymes that hydrolyze the internuclear linkages in phosphodiester bonds
32
What are polymerases
catalyze template dependent synthesis of DNA
33
What are ligases
enzymes thst create phosphodiester linkages
34
What are the 3 major domains of DNA pol I?
The DNA polymerase
the 3'-5' proofreading exonuclease and the 5' to 3' exonuclease
35
How does DNA pol I seal nicks?
at a nick in the DNA, pol I degrades the RNA primer in the 5' to 3' direction releasing dNMPs and simultaneously extends the 3' terminus with dNTPs, removing RNA and replacing with DNA
36
What does the structure of DNA pol I resemble?
A right Hand with three separate domaines.
Figer thumb and palm
37
What is the fingers domain?
where the dNTPs enter
38
What is the thumb domain?
Holds the template DNA strand in place so it can incorporate nucleotides without disruption
39
What is the palm domain?
DNA template rests here and contains the active site
40
What are the two conformations of DNA polymerase?
open and closed. undergoes this change for every dNTP in corporated
41
What is the open form?
free 3; OH of the strand synthesized is sitting in the active site and finger has contacted a dNTP
42
What is the closed form?
dNTP has to be in proximity to the growing strand and attached at the insertion site. closed form accomplishes this by bringing the finger domain 40 degrees closer to the active site
43
How does the polymerase incorporate the correctly paired nucleotide into the growing strand?
relies on shape recognition involving H-bonding, van Der Waals, and ionic bonding interactions. AT and GC bps have shapes that fit well into the closed form of the active site
44
How much slower is the incorporation of incorrect bases?
10 to 1000 fold slower than correct bases. further, correct base pairing and base stacking of the newly added NT provides additional stabilization that favours incorporation of the correct base
45
What does an incorrect fit result in?
dissociation of the dNTP and binding of a new one.one in every 10^4-10^5 nucleotides is added incorrectly
46
How many conserved components are there in the polymerase
3
47
What are asp residues?
once the correct fit is found, the reaction proceeds using two essential asp residies coordinating with two divalent Mg++ ions. Mg plays a direct role in catalysis
48
What is the first magnesium ion?
The second conserved component. Deprotonates the 3'OH to generate a 3'O- Nu which can attack the alpha P on the incoming dNTP
49
What is the second magnesium ion?
responsible for binding the negatively charged phosphate groups on the incoming dNTP and facilitating the rapid release of PPi
50
What is the first step of DNA polymerase reaction?
DNA requires several componenets/ Primer strand and template strand as well as dNTPs
51
What is the second step of the DNA polymerase reaction?
dNMP is incorporating and polI will move forward to the new 3' terminus to incorporate another dNTP. There is a post insertion site and an insertion site
52
How is the polymerase able to slide?
several positively charged lys residues in the palm interact with negative phopshodiester backbone via electrostatic interaction
53
Why does polymerase have processivity?
it is able to catalyze consecutive reaction without releasing its substrate due to the strength of the lys backbon interaction
54
What is processivity number?
Shows the effectivity of polymerases. low processivity is like pol I whereas polII has a high number
55
When will DNA pol add to the previous addition?
only if the previously added base is firmly paired
56
Where is the 3' to 5' exonuclease domain on DNA pol?
side of palm on fingers side
57
How does the 3' to 5' exonuclease activity of DNA pol work?
dsDNA will be frayed by 4 nt (3 correct 1 incorrect). H bonds between bp are disrupted and 3' to 5' exonuclease will cleave the incorrect dNMP from the growing polypeptide chain
58
What does 3' to 5' exonuclease use to catalyze the removal of the incorrect dNMP?
with two metal ions
59
What is the function of the first Mg2+ in the 3'5' exonuclease
deprotonates H2O molecule to make OH Nu which accacks phosphate and mediates hydrolysis of dNMP leaving 3' OH
60
What does the other Mg ion in 3'5' exonuclease do?
promotes departure of dNMP LG from the growing strand
61
What does DNA pol I also have in addition to 3'5' exonuclease?
5'3'exonuclease. This degrades RNA or DNA while polymerase dimultanesouly adds. EX. in the Okazaki fragments
62
Describe the process of nick translation in pol I?
at nick, pol I degrades RNA primer in 5' to 3' direction
releases dNMPs and extends 3' terminus with dNTPs in same direction
net result is movement of the nick in the 5' to 5' direction along DNA until all RNA is gone
DNA ligase can seal fragments following pol 1 dissociation
63
Descibre E coli pol I
Okazaki fragment processing ad DNA repair with 3-5' and 5-3' exonuclease activity
64
Describe polII?
translesio synthesis with 3-'5 exonuclease activity1
65
Describe pol III?
chromosome replication with 3-5' exonuclease activity
66
Describe pol IV and V?
translesion synthesis with no exonuclease activity
67
What are the three main stages of E coli replication?
initiation of replication, elongation by the replisome, ad termination
68
What is OriC>
the single origin of replication in E coli that is 245bp long containing A-T rich 13-mer repeats ad DnaA 9-mer sites
69
What are DnaA 9-mer sites?
four copies of a 9 nt consensus sequence to which the bacterial initiator protein binds
70
What are A=T rich 13-mer reapeats?
3 A=T rich direct repeats (same directionality) of 13bp each. Referred to as the DNA unwinding element, these unwind readily upon binding of the initiator
71
What are the steps of the activation of OriC?
generation of the open complex, activation of the origin of replication, assembly of the E. coli replication forks, replication initiation and leading strand synthesis, lagging strand synthesis
72
What I\s the initiator protein of the E coli open complex?
DnaA. A member of the AAA+ protein family, which bind and hydrolyze ATP
73
What occurs when
74
DnaA binds to OriC?
DnaA oligomerizes nd wrap the DNA round the oligomer like a scarf, putting strain on the DNA. Destabilizes AT using ATP at the 13mer repeats.
75
What is the open complex?
the complex of DnaA-ATP-OriC-HU (basic histone like protein)
76
How is the replication origin activated?
two hexamers of DnaB assemble on each ss. DanC (helicase loading protein) loads DnaB and represses its helicase activity when ATP bound. When hydrolyzed, it releases and DnaB is free to act
77
How its the E coli replication fork assembled?
DnaB binds ATP enabling It to translocate and unwind DNA, dislodging DnaA. unwinding in both directions generates supercooling stress which is relieved by topoisomerase. unwound DNA is also bound by SSB proteins
78
How big must the bubble be for primate to interact with DnaB
100-200 base pairs
79
What occurs during replication initiation and leading strand synthesis?
primer directs loading of the B-clamp and assembly of the leading strand pol II holoenzyme
80
What is a holoenzyme?
The active form of an enzyme that is bound to its cofactor
81
What is an apoenzyme?
an inactive enzyme that is not bound to the cofactor
82
What is the B -clamp
a ring shaped homodimer that slides along the duplex DNA ahead of the pol III core to increase processivity
83
What is lagging strand synthesis in E coli
each pol III holoenzyme extends it RNA primer until both pol complexes connect with the DnaB helicase travelling on the other side of the replication bubble =. Priming is followed by clamp loading and synthesis of the lagging strand by a second pol III.
84
How many origins of replication do eukaryotes have
many
85
How far apart ore origins of replication spaced in eukaryotes?
10-40kbp apart along each chromosome
86
How many times
87
how many times do origins fire per cell cycle
once
88
What are ARS is S cerevisiae?
replication origins 100-220bp long with a highly conserved A sequence and AT rich B1, B2 and B3 elemtns
89
What is the eukaryotic initiatior?
heterohexamer called ORC containing subunits with actions similar to DnaA. ATP required for binding to the origin
90
What occurs after ORC binding to DNA
Cdc6 protein (AAA+) also binds to ORC. complex then loads Mcm2-7 onto DNA
91
What is MCM2-7?
A circular helicase hetwerohexamer the binds one molecule of Cdt1 (DnaC like) before being loaded by the ORC-Cdc6 protein onto the DNA
92
What is preRC?
pre replication complex containing ORC, Cdc6, Cdt1, and MCM2-7
93
What is the replisome?
A multiprotein complex that promotes DNA synthesis at the replication fork. Composed of Pol III holoenzyme,, DnaB helicase, and primase
94
What is the polIII holoenzyme?
A 22 subunit DNA polymerase III complex found in E coli responsible for DNA replication. Consists of III B-sliding clamps and a clamp loading complex and 3 pol III cores
95
What is the enzyme that opens the replication fork?
DnaB
96
Where does DnaV connect other the replisome?
through the tau subunits of the clamp loader p\
97
Why is DnaB connected to the polymerase?
It has low processivity (35bp/s) without connevtion and high processivity with connection (700bp/s)
98
How many pol II cores are associated with the lagging and the leading strand?
1 with the leading and two with the lagging
99
What terminus of the tau subunit protrudes from the clamp loader and binds the pol III cores?
The C temrini
100
What is the structure of the B clamp?
homodimers shaped like a ring encircling the DNA duplex
101
What is the function of the B clamp?
To hold the polymerase onto the DNA while sliding along the duplex. Converts pol III from a distributive enzyme to a processive enzyme
102
What is the structure of the pol II core?
similarity to pol I. Palm, thin, and finger
103
What is the function of pol III?
alpha subunit is the main replicative subunit and it recruits the tau subbing for 3-5 proofreading
104
What do B clamp facilitate?
Processivity. 1kb/s and 100kb per binding event
105
What is the purpose for 3 pol III cores?
facilitates coordinated synthesis of the leading and the lagging strands
106
How many clamp loaders are needed to assemble the clamps to both DNA strands?
one
107
How is the holoenzyme held together?
weak interaction including H bonds and van Der Waals forces
108
What experiment was conducted to provide evidence for B clamp function?
a purified pol II holoenzyme with one B clamp and one pol II core is assembled onto a primed donor ssDNA circle and then mixed with competeing acceptor DNA circles of different sizes Each contained a primer but only one included a clamp
109
What was reaction B?
smaller DNA has a B clamp and the other does not
110
What was reaction A?
bigger has a clamp and smaller does not
111
How was replication initiated in the experiment?
radioactive 32P dNTPS
112
How were the results analyzed?
aliquots of the reaction mixture are analyzed on an agarose gel followed by autoradiography. Both cases, the donor plaid was replicated first, followed by plasmid with the clamp
113
What is the clamp loader composed of?
the tau complex (tau trimer, delta, and delta prime subunits)
114
How does the clamp loader load the clamp?
ATP binding to tau subunits induces a conformational change that allows the binding and opening of the clamp
binding of ATP is needed for the clamp loader to bind DNA
ATP hydrolysis causes he clamp loader to revert to the form that cannot bind the B clamp or DNA, ejecting the clamp loader and allowing the clamp to land ar0und the DNA
115
What is the trombone model?
A model used to describe replication on the lagging strand with repeated cycles of loop growth and disassembly which is analogous to the movement of a slide on the trombone
116
Why does a loop form with Okazaki fragments?
Because DNA synthesis opposes the movement of the replication fork
117
Who occurs once the Okazaki fragment is synthesized?
The DNA is released and the polymerases dissociates from the DNA (detatvches from the B clamp) and attaches to a new primed site with a B clamp (buildup of primers and clamps)
118
What are the four steps of the resolution of B clamp buildup?
B clamp sites attract pol I which removes RNA primer
Pol I dissociates leaving an ss DNA break. Attracts DNA ligase which seals the break
unoccupied B clamp is then opened and unloaded by excess delta subunits of the clamp loader
119
What are Ter sites?
two clusters of the 23 bp sequences that are oriented in opposite directions
120
What is Tus?
termination utilisation substance that binds to her and blocks the advance of the replication fork by stopping the DnaB helicase
121
How are Tus-Ter complexes directional?
holoenzyme can pass one way and not the other way
122
What occurs when replication forks are of equal speed?
They meet at the same time in the terminus region
123
What happens when replication forks are of unequal speed?
Meet in the terminus region
124
What occurs with codirection collision between RNA pol and DNA pol?
they do not impede forks
125
What occurs with head on collision between RNA pol and DNA pol?
can cause the fork to stall or pause
126
How are most transcripts oriented in bacteriA>
Same direction as replication
127
Why may have the ouster system evolved?
To prevent replication forks from going too far around the chromosome, causing head on collision
128
How ia catenation resolved?
by action of a type II topoisomerase
129
Describe type II topoisomerase?>
It is a heterodimer consisting of two ATPase domains and two cleavage cores domains linked by a scaffolding domain.
130
What is the mechanism of type II topoisomerase?
enzyme pulls of of the pieces of catenated dsDNA into its cleavage site and through the use of ATP it can generate as ds break in the DNA fragment
131
What is the (detailed) mechanism of type II toposisomerase>?
tyr residue in the catalytic site which gives up its proton to form a nucleophile which attacks the phosphate in a phosphodiester bond. Links DNA and protein, forming a phosphtyrosyl linkage known as a 5' adduct. Once separated, hydrolysis reforms phosphodiester
132
What is the problem with linear chromosomes?
Once the RNA primer at the extreme end is removed for replacement there is no 3' terminus for pol to attach , leaving a gap. Replication will repeated shorter this gap and genetic info will be lost
133
What are telomeres?
repeating sequences at the end of chormosomes
134
What is TERT?
telomerase reverse transcript which carries tightly bound non coding telomerase NA (TR) which contain 1.5 telomere repeat units which match the telomere repeat sequence that it uses as a template to extend the 3' terminus of the telomere beyond what was replicated
135
When does telomere extension occur?
S phase
136
Descibe the reaction cycle of tetrahymena telomerase
at 3' terminal end of linear DNA, 3 its of the telomere anneal to 3 RNA nt in the telomerase
telomerase extends the 3' end of ss DNA by the length of one telomere repeat (6 nt for this organism)
after adding repeat, telomerase separates the RNA DNA hybrid and repositions on the telomere for extortion of next 6-mer repeat. Telomerase is processive
telomere extends a 3' singl stranded DNA teminus is then converted to duplex DNA by same primeing and polymerization machinery used in chromosome replication. Because telomere is intronic, there can be variability in final lenght
final terminus is not completely duplec
137
How is telomerase different from other DNA pol?
carries its own template and synthesizes ssDNA
138
What is a karyotype?
A visualization of the number and appearance of the chromosomes within a cell where each chromosome pair is labelled with a different colour
139
Where do problems within a cancerous cell arise from?
result of faulty recombination and DNA repair
140
What is aneuploidy?
The presence of an abnormal number of chromosomes in a cell
141
What are chromosomal translocation?
rearrangement of parts between non-homologous chromosomes. Generally caused by errors during homologous recombination or ds break repair
142
What is a double strand break?
a break in the phosphodiester backbone on both strands of DNA at the same site. Usually arise during replication when the replication fork encounters a single strand break in the template strand which is then propagated into a break in both the mother and the daughter strands. Can also result from UV or gamma radiation. Leads to cell death if not repaired
143
What is deinococcus radiodurans?
A bacteria that can withstand large amounts of radiation. Regenerates its DNA every 12-24 hours despite DSBs
144
Describe the D. radiodurans genome
four circular DNA molecules, present in multiple copies (backup)
145
What occurs upon irradiation of D. radiodurans with y radiation?
cell stops growing and overlapping DNA fragments are spliced together and the entire genome is accurately reconstituted within a few hours before cell growth is resumed
146
What is recombinational DNA repair?
directed at the repair of DSBs. or cross-links especially at inactivated replication forks. Requires the presence of another undamaged homologous dsDNA. In diploid cells, this is the second copy of the chromosome or the sister chromatid present after replciation
147
What is the process of recombination DNA repair (steps)?
First, broken DNA ends are processed by helices and nucleases with the 5' ending strands selectively degraded to create 3' ss extensions . ss DNA is coated and protected by SSBs
3' overhangs invade homologous chromosome, catalyzed by recombinases (reca proteins) which replace the SSBs. Displaces one strand of the intact homologous chromosome and base pairs with the other. The structure created by strand invasion by 3' single stranded extension is sometimes referred to as a D loop
Second invasion takes place
replicative extension of the invading strand by DNA pols
148
What are the two pathways to complete repair of DSB?
Synthesis dependent strand annealing
DSB repair
149
What is synthesis dependent strand annealing?
now lengthened strands can simply be displaced by the action of helicases and then anneal to each other. remain gaps filled by DNA pols . ligases ligate
150
What is DSBR?
ligation. of strand while they are still linked, creating a four branched cross-over junction. Creates holiday intermediates which are resolved by special endonucelases called holiday junction resolvases
151
What happens if both holiday intermediates are cleaved at X sites or Y sites?
non-crossover, which means genetic material will be exchanged between cleavage sites, but not between the chormosomes
152
What occurs in XxY resokution
genetic material outside repair is from two separate chromosomes
153
Which polymerase Is involved in bacterial recominatorional DNA repair?
pol I
154
How did homologous recombination begin?
As a DNA repair process but evolved into a broader evolutionary mechanism
155
What is homologous recombination?
recombination between two DNA molecules of similar or very similar sequences during meiosis and mitosis in eukaryotes and during DSB repair in all organisms
156
What ensures the segregated chromosomes are properly linked in meiosis I?
homologous recombination which provide accurate alignment of homologous chromosomes at the metaphase plate during meiosis
157
What enzymes are involved in meiotic recombination?
same ones involved in recombinatorial DNA repair
158
What is the second role of crossing over?
to exchange parts of each set of paired chromatids in order to create genetic diverstiy
159
160
Describe the first stage of meiosis?
homologous chromosomes are aligned and the crossovers of recombination are visible in prophase I. Homologous chromosomes partially separate but are still held together at joints called chiasmata where the actual recombination occurs
161
Describe the initiation of meiotic recombination in yeast cells
DSBs are intentionally introduced at multiple locations along one chromatid of each chromatid pair . Breaks are not random but not predictable/ Breakage occurs at chromosomal hotspots by a protein called spo11
162
How is spo11 removed from DNA?
by a complex of proteins (Mre11-Rad50-Xrs2) which bind to each Spo11 complex, valeting the DNA by several basepairs on the 3' side, liberating the protein along with a short segment of the attached DNA strand
163
What occurs after liberation of Spo11?
nuclease, Sae2 then degrades the DNA more, and other enzymes like helicase Sgs1 and nucleases Exo1 or Dna2 are implicated in the more extended degradation of the 5' ends to create long 3' ss extension which can now be processed in a mechanisms similarly to DSB repair
164
Descibre the recombination DSBR pathway
ss regions bound by RPA (eukaryote SSB). mediator proteins aid in the addition of two RecA class recombinases called Dmc1 (disrupted meiotic cDNA) and Rad51 are loaded onto the 3' extensions on either side of the ds break.
binding, sampling and release until Watson crick base pairing allows for homology
stable DNA joint is then formed by intertwining of the ssDNA with its complement form the homologous target
165
What is RubAB?
Repair of UV damage. complex. two RuvB hexers and two RuvA protein tetramers. bind to a Holliday intermediate
166
What is RuvA?
a Holliday junction specific DNA binding protein that recognizes the structure of the DNA junction and keeps it in a box-like state
167
What is RuvB?
Donut shaped hexamers surround two of the 4 arms of the Holliday intermediate. DNA translocate, similar to hexametric DNA helices. DNA propelled outward through the hole in the protein, in a reaction coupled with ATP hydrolysis/ Rapid movement of the position of the intermediate
168
What is RuvC?
holiday intermediate resolves which replaces one of the RuvA tetramers at the junction and cleaves errands in opposing arms of the Holliday intermediate to resolve it into viable chromosomal products . Nicks two strands with the same polarity. Nicks are later sealed into a different arrangement by DNA ligase
169
What is site specific recombination?
A precisem, predictable process in which DNA is rearranged between two specific sequences. involves movement of mobile genetic elements between non-homologous sites. Carried out by recombinases and can result in in insertion, deletion or inversion of a particular DNA segment.
170
What are the effects of site-specific recombination?
can alter gene order (wouldn't happen during homologous recombination due to exchange of same parts). Used by prokaryotes and eukaryotes to regulate gene expression and to increase and organisms genetic repertoire
can cause spontaneous mutations in organisms.
relics of site specific recombination are found in many vertebrae organisms (~45% of the human genome [higher in plants])
171
What can be produced with site specific recombination?
transgenic organisms using recombinase enzymes
172
How are recombinate systems used in a biotechnology capacity?
to activate a gene, inset a gene, replace a gene, delete a gene, or alter the linear strucutre of a chromosomes
good for knock-in/knock-out mice
173
What two systems are used in site-specific recombination for genome editing?
Cre-Loc and Flp-FRT
174
What are LoxP and FRT?
specific directional sequences that are placed into the genome (not naturally occuring
175
What are Cre and Flo?
recombinase enzymes that recognize LoxP and FRT sites respectively. Not naturally occurring in eukaryotes but work to cleave or invert the intervening sequence when enrgineered into the cells of any organism
176
How many LoxP or FRT sites must their be for the system two work?
two
177
What occurs if the sites are facing one another (inverted)
the enzyme will invert the intervening sequence (change orientation on. the DNA)
178
What occurs if the sites are oriented in the same direction?
cleavage of the intervening site into a plasmid
if a foreign piece of DNA containing homologue to the region outside of the LoxP or FRT sites and also contains two matching sites the intervening sequence of the foreign DNA can be inserted, replacing the endogenous sequence in between the sites
179
What is the brainbow mouse?
individual neurons in the brain are visualized using fluorescent proteins using transgenic tech and site specific recombination
180
What is the rainbow technique?
gene casette inserts din mous genome with several GFP variants. LoxP variants are engineered between them
different lox sites have different structures so lox1 only reacts with lox1 etc...
Are mediated recombination results in different patterns of GFP variant expression in each neuron. Sites are arranged so only one of three recombinations can occur in a cassette and each event will result in gene expression of one of four color variants
promoter in cassette that directs gene expression in neurons of only most proximal GFP variant. mice have been engineered with several cassettes. transgenic mice are homozygous for the casettes/ Other mice are engineered expressing cre recombinase
181
What is the outcome of rainbow?
GFP mouse and Cre mouse mate and progeny show fluorescence in neurons
182
How is gene function often understood?
deletion (often through cutting a gene at a critical site producing DSB
183
What three technologies have been developed to cut genes at particular sites in vivo?
zinc finger nucleases
TALENs
CRISPR/Cas systems
184
What are zinc finger nucleases?
a designer DNA cleavage system based on using a zinc finger containing protein to recognize a specific site and cleave it
185
What is a zinc finger?
protein domain characterized by a single atom of zinc coordinated to four cysteine residues. 30aas and folds into a characteristic B-a-B structure bound with a. Zn2+ ion
186
How can a zinc finger contact its within the major groove of DNA
using amino acid residues within the alpha helix structure of the zinc finger. binds with a substantial degree of selectivity
187
What are the applications of ZFNs?
zinc fingers can be stitched in tandem to create structures recognizing NDA sequences 9-18 bp long.
188
Where do zinc fingers cleave
non-specific nuclease domain (often derived from an enzyme called Fokl) cleave adjacent to the recognition site of the associated zinc fingers
189
What are TALEs?
Trancription activator like effector domains that direct DNA binding in TALENs
190
Describe TALENs?
similar in size to zinc fingers but recognize single base pairs. Can be linked to gather and fused to a non-specific nuclease domain. Can be expressed in a cell and cleave the target site to generate a DSB
191
What are TALENs and ZFNs designed to do?
inactivate genes or integrate specific foreign DNA sequences. Can even inactivate viral DNA in the genoe
192
What are the limitation of ZFNs and TALENs?
they require modularity to recognize different DNA sequences. Different designer enzyme for each sequence you want to recognize Requires much expensive protein engineering
193
What is CRISPR/Cas?
a gene cutting approach derived from bacterial immune system standing for clustered regularly interspaced short palindromic repeats and crisps associated proteins
194
Describe CRISPR/Cas in bacterial immune systems?
viral DNA seized by the bacterial immune system, is incorporated into CRISPR regions to become spacers
expressed as short RNA molecules which can form larger seek and destroy structures when conjoined with the Cas9 nuclease
protects cell and records previous viral attacks
195
Describe the CRISPR/Cas mechanism?
CRISPR sequences are transcribed into RNA and spacers which are cleaved to form products called guide RNAs . gRNA complexes with the cas protein and often a trans-activating CRISPR RNA. RNA folds into a hairpin loop which is recognized by Cas. resulting complex binds specifically to invading bacteriophage DNA cleaving it
196
What was developed in Jennifer Doudna and Emmanuelle Charpentier's laboratories?
guide RNA and trans activating CRISPR RNA are typically fused into a single guide RNA that does not exist in the endogenous bacterial system. Can be programmed to target almost any specific genomic site
197
Describe the nuclease domains of Cas9?
one domain that cleaves the DNA strand paired with the sgRNA and the other cleaves the opposite strand
198
What occurs If one nuclease domain in CAs9 is inactivated?
enzyme nicks one strand, causing repair using homology directed repair
copy paste mechanism where DNA sequences similar to those around DNA break site (introduced by researcher) act as a repair template
199
What occurs if a DSB is introduced with CAs9?
non-homologous end joint which joins loose ends. error prone and often disrupts the gene
200
How is inactivation carried out with CRISPR/Cas9?
electroporation and gene knockout occurs
201
How are genomic changes introduced with CRISPR/Cas9?
introduced using recombination using a DNA fragment encompassing a cleavage site and including the planned change
202
What are the six domains of the Cas9 protein?
Rec I, Rec II, Bridge Helix, RuvC, HNH (His-Asn-His) and PAM-InteractiongW
203
What is the PAM sequence?
A NGG trinucleotide sequence where N is any base. Always found next to spacer sequences and is necessary for Cas9 to be able to bind target DNA. Must immediately follow the target sequence and distinguishes invading DNA from self-DNA
204
Describe the endogenous CRISPR/Cas system?
In bacteria, mature crRNA base pairs to trans-activating crRNA forming a dual RNA structure that directs the CRISPR associated protein Cas9 to introduce DSBs in DNA adjacent to PAM.
At sites complementary to crRNA guide sequence, HNH nuclease domain cleaves the complementary strand and the Cas9 RuvC-like domain cleaves the non-complementary strand
205
BCHM 218
flashcards
Decks in class (2)
# Cards
