1
Q
The storage of glucose-6-p costs what?
A
1 ATP
2
Q
How does insulin respond to blood glucose levels?
A
Insulin is released into blood from pancreatic beta cells when there is lots of glucose in the blood
3
Q
What causes the secretion of insulin?
A
-glucose enter b-cell via glut 2–phosphorylated to G6P and metabolized–increase ATP–ATP closes K+ channel–voltage gated Ca2+ channel opens increasing Ca2+–insulin secretion increases–transciption of insulin gens
4
Q
How does glut 2 facilitate glucose uptake?
A
Glut 2 is is low affinity for Gluc, can only bring in gluc when a high concentration outside.
5
Q
What causes the Ca2+ channel to open?
A
Increase of ATP from influx of Gluc and the metabolism of G6P causes more ATP to be produced. ATP inhibits K+channel causing the cell to depolarize.
6
Q
How does glucagon respond t blood glucose levels?
A
Glucagon is released into blood from pancreatic alpha cells when there is little glucose in the blood?
7
Q
What is glucagon?
A
- polypeptide hormone
- made from alpha cells
- released when blood glucose low
- mech of stimulation is similar to insulin, but not yet known
8
Q
What affect those endurance exercise have on glucose, glucagon, and insulin levels?
A
- increase glucagon
- decrease in glucose levels
- decrease in insulin levels
9
Q
What happens when insulin binds to a dimer of a single-transmembrane receptor?
A
Insulin binds to the alpha subunit of the receptor, causing a cascade of kinases that ultimately activates protein phosphatase-1
10
Q
What is the difference between a Epinephrine structure and a glucagon structure?
A
- Glucagon is a polypeptide of amino acids
- Epinephrine is a cathecolamine, which is aromatic ring connected to and amine group
11
Q
What do hormones like epinephrine or glucagon do?
A
-activated adenylyl cyclase by binding to G-protein coupled receptors (GCPRs).
12
Q
How does GCPR work?
A
(7 transmembrane helices)
- Heteromeric G protein is bound by a hormone in the Adrenergic receptors
- GDP dissociates from GCPR and is replaced by GTP
- structure dissociates to adenylate cyclase turning ATP to Cyclic AMP Activating protein kinase A.
13
Q
How is the response to a hormone terminated?
A
- GTP bound to the G-protein is hydrolyzed to GDP
- Either the hormone dissociates form the receptor
- or the receptor becomes phosphorylated
- Arrestin binds to the phosphorylated receptor to cap it (dissociating hormone)
14
Q
What drives the reaction of ATP to cAMP to 5’AMP?
A
The conversion of P2 to SP.
15
Q
What enzyme turns ATP to cAMP?
A
adenlyate cyclase (AC)
16
Q
Which enzyme turns cAMP to 5’AMP
A
phosphodiesterase
PDE
17
Q
What does caffeine do?
A
It inhibits PDE and increases cAMP levels into the body
18
Q
Epinephrine and norepinephrine attaches to what receptor to activate AC?
A
Beta adrenergic receptor of muscle and liver cell
19
Q
Glucagon attaches to what receptor to activate AC?
A
Glucagon receptor in the liver only
20
Q
What are the subunits of PKA?
A
R2- regulatory subunit (inhibits C)
C2- catalytic subunit
21
Q
What activates PKA?
A
cAMP binding to R2 of PKA, releasing the 2 C subunits.
22
Q
How is glycogen breakdown activated?
A
cAMP activates PKA–PKA uses ATP to phosphorlyze phosphorylase kinase b to kinase a–phosphorylase kinase a gives glycogen phosphorylase b a phosphate turning it into a–glycogen turned to glucose-1-P.
23
Q
Glycogen Phosphorylase b favors which conformation more?
A
favors T state more
24
Q
Glycogen phosphorylase a favors which conformation more?
A
favors conformation R
25
Why is T state of Glycogen phosphorylase less active
active site is buried
26
What s Mcardle's disease?
- Muscle glycogen phosphorylase deficiency
- Exercise intolerance, muscle pain, fatigue, cramps
- 100 mutations in genes
27
What is Hers" disease?
- Liver glycogen phosphorylase deficiency
- Enlarged liver, hypoglycemia, KB
- 17 mutations in the gene
28
How is Glycogen phosphorylase allosterically in Muscle?
- responds to energy of the cell
- Epinephrine pushes it to a, even without amp
- ATP activates it
29
How is Glycogen phosphorylase allosterically in Liver cells?
- responds to glucose
| - insulin can deactivate
30
What does phosphorylase kinase do?
phosphorylates phosphorylase
31
What is the structure of phosphorylase kinase?
- - huge enzyme complex of ~ 1,200 kDa
| - - consists of 4 different subunits, each is present 4 times: (α, β, γ, δ)4
32
What are the functions of the phosphorylase kinase subunits?
- γ = catalytic subunit, has an auto-inhibitory C-terminus
- α, β, δ = regulatory subunits
- α and β subunits: both are inhibitors. Once they are phosphorylated, they move away from the γ-subunit.
- δ = calmodulin (CaM = Ca2+ binding protein); “de-inhibits” the γ subunit when Ca2+ is bound.
33
For maximum activity of phosphorylase kinase, what is need?
-calcium and ATP
34
What is phosphorylase kinase kinase?
cyclic AMP dependent protein kinase
| = PKA (has many other substrates)
35
What is phosphorylase kinase phosphatase?
phosphatase-1
36
How is glycogen synthase regulated?
-phosphatase 1 inhibits, to be full active needs G-6-P
-- important glycogen synthase kinases are phosphorylase kinase
and cAMP dependent protein kinase
37
How does insulin reverse effects of glucagon and epinephrine?
reverses effects of glucagon and epinephrine by activating
a protein phosphatase (PHOSPHATASE-1) that
dephosphorylates phosphorylase kinase, phosphorylase
and glycogen synthase.
38
What is insulin?
- peptide hormone
- binds to insuline receptor tyrosine kinase in liver, muscle, adipose
- produced by bets cells in pancreas when glucose high
- stimulates glucose uptake in muscle and adipose
39
Can muscles store more energy in glycogen than liver cells?
yes, about 4 times more
40
What are the phases of starvation?
1. Glycogenolytic: glycogen to glucose
2. Gluconeogenic: AA to glucose
3. Ketogenic: FA to KB
4. Terminal: AA used up from remaining protein
41
What occurs in the glycogenolytic phase?
- Liver exports glucose and glycogen is rapidly depleted from the liver
- other cells lack G-6-P so they cannot release glucose
42
What occurs in the gluconeogenic phase?
-glycogen gone, and brain still needs a lot of glucose
-FAs can be used to make glucose
-sources of glucose left:
– Glycerol from TAG hydrolysis
– Amino acids from protein.
– Odd chain fatty acids
43
Gluconeogenesis requires how much energy?
6 ATP
44
What adaptation occurs in the gluconeogenic phase?
-Gluconeogenic capacity increases over time of phase
45
What is the problem of the gluconeogenic phase?
- proteins are degraded too fast
- Humans cannot survive lost of 1/3 protein.
- last only 20-30 days, can fast 40-50 though
46
What occurs in the ketogenic phase?
- use energy stored fats
- KB production increases
- brain adapts to KB
- glucose consumed
- less AA breakdown
47
What occurs during the terminal phase?
- Run out of fat
- Start degrading massive amount of protein
- Pe morbid, urea increase
- Death
48
What increases plasma glucose?
- diet
- gluconeogenesis (liver, kidney)
- glycogen (liver)
49
What decreases plasma glucose?
- insulin
- Tissue uptake (muscle, adipose)
- oxidation (CNS, all tissues)
50
Glucose level of 1.1/20 indicates?
coma
51
Sx of hypoglycemia are indicated at what plasma glucose level?
2.2/40
52
Fasting rage glucose levels are at wat?
3.3/60
53
Normal post-adsorptive/euglycemia are at what levels?
5.5/100
54
Why are high of glucose bad?
reactive aldehyde group reacts with glucose and inactivates proteins.
neurons, kidney, retina
55
What occurs to plasma glucose levels with people with type II diabetes?
-Can still make insulin but cells are not responsive
56
What occurs in IDDM?
- - lack of pancreatic β-cells, no production of insulin -- cause: autoimmune disease
- - treatment: insulin, diet (frequent small meals)
57
What occurs with NIDDM?
-- body is resistant against insulin
-- risk factors: obesity and genetic predisposition -- treatment: change of lifestyle (diet, exercise)
insulin
drugs: to inhibit gluconeogenesis in liver
to increase glucose uptake by muscle
58
What are the consequences of a lack of insulin?
1. Impaired glucose uptake by muscle and adipose tissue- high glucose levels in blood.
2. Glucagon prevails (starvation in the presence of high glucose)
→ gluconeogenesis active
→ high levels of fatty acids and ketone bodies
59
Why are increased glucose levels so damaging?
- - cardiovascular problems, cataracts, blindness (remember that glucose has reactive aldehyde group)
- - excretion of glucose by kidneys: dehydration (thirst is diagnostic indication), kidney failure
60
Why are high levels of KB damaging?
-- cardiovascular problems, ketosis (acetone smell is diagnostic indication). Ketosis can lead to coma, low pH leads to
kidney failure as protons are constantly excreted.
61
What is Diabetic Ketoacidosis?
-No insulin is made, lots of KB in blood, pH falls, coma, then death
62
How does glipizide (glucotrol) help type II DM?
Blocks pancreatic beta cells K+ channels, stimulates insulin secretion by pancreas.
63
How does GLP-1 modulaters treat DM type II?
Targets Glucagon-like peptide 1 with dipeptide protease IV, Enhances insulin secretion by the pancreas.
64
What are the effects of incretins?
• Incretin effect: oral glucose elicits a stronger insulin response than equivalent IV challenge.
• Due to release of hormones from neuroendocrine cells of intestine (L-cells) that modulate insulin release
(GIP and GLP-1)
65
What is the purpose of GLP-1?
– Binds to GPCR on β-cells and activates a G-protein.
– Enhances insulin secretion from β cells.
– Decreases glucagon release from α-cells.
– Enhances β-cell proliferation and increases cell mass.
66
What activates GIP and GLP-1?
DPP-4
67
What is DPP-4?
– Expressed on the surface of most cells
– DPP-4 inhibitors increase GLP-1 and enhance insulin release.
– first DPP-4 inhibitor was isolated from saliva of Gila monster.
68
What is the affect of GLP-1 and CKK on the brain?
- increased satiety
- decreased food intake
- decreased body weight
69
What does leptin do?
- inhibits eating behaviors and slows fat synthesis.
- Stimulates FA oxidation
- the more fat, the more leptin in blood
70
What is Ghrelin?
Produced by cells in stomach and pancreas when food levels in stomach are low
71
How is our appetite regulated by peptide hormones?
Ghrelin: “I’m hungry” - hormone, secreted by empty stomach
Leptin: “Thank you I am fine” - hormone, secreted by adipocytes -- dietary fatty acids induce secretion
-- basal levels correlate with mass of fat
72
Aside for precursors for RNA/DNA, what are some other purposes for nucleotides?
Energy, substrates, phosphorylation, signaling coenzyme factors
73
List all the nucleotides
ATP, GTP, cAMP, UDP-glucose, GDP, NADH, FAD, SAM
74
What distinguishes RNA from DNA?
The sugar
75
What are tautomers?
structural isomers that differ in the position of protons and double bonds. Can lead to unusual base pairing and rarely mutations.
76
What is the difference between nucleosides and nucleotides?
the 5'-pohosphate
77
What makes RNA less stable than DNA and gives it its catalytic properties?
2-OH'
78
What three components are necessary for both Purine and Pyrimidine ring synthesis?
HCO3-, Gln, Asp
79
What is unique to the synthesis of purine rings?
Gly, Formyl-THF
80
What is PRPP?
an important intermediate in Purine and Pyrimidine synthesis and salvage pathways. - Activated ribose-5’-P donor
81
What is PRA?
First piece of the purine ring
82
What is the committed state of purine synthesis?
production of PRA
83
What drives the production of PRA?
pyrophosphate
84
What is a key difference in purine synthesis that pyrimidines?
They are built up stepwise from a ribose platform. Pyrimidine is built from the ring.
85
How many more steps from PRA does it take to make IMP?
9, cost 4-5 ATP, need a lot of AA
86
What are is so important about enzymes in purine synthesis?
```
Enzymes of Purine biosynthesis
• Organized for substrate
channeling
• Protect labile intermediates
• Enhance “low” concentrations
• Joint Regulation of enzyme
activities
```
87
What is a purinosome?
Enzyme activities required to convert PRPP to IMP plus Ser-Hydroxy-Methyl Transferase (SHMT) & C1-THF Synthase.
Localizes protein and purines.
88
IMP can be converted to make what two compounds?
XMP or adenyl-succinate (GMP or AMP)
89
What allows GMP and AMP to be made from IMP?
GMP- Gln + ATP
| AMP- aspartate, GTP
90
What is the key to the balance of nucleotide sythesis?
1. [GTP] drives AMP synthesis [ATP] drives GMP synthesis
2. AMP and GMP regulate their own synthesis from IMP
3. Gln-PRPP Amido-transferase
Binds AMP and GMP at different sites. Concerted allosteric regulation
91
What inhibits Glutamine-PRPP amidotransferase?
IMP,GMP,AMP
92
What are the three sites of CPS-II?
glutamine amidotransferase, synthesis of carbamate, synthesis of carbamoyl-P
93
How is pyrimidine synthesis regulated?
-Substrate Channeling in Pyrimidine Synthesis:
CPSII, ATCase, DHOase on one 210kD polypeptide chain (CAD)
-ATP and PRPP activate CPSII (substrate activation)
-UDP and UTP inhibit CPSII (product inhibition)
94
How is CTP made?
UMP is converted to UTP, then CTP by CTP synthase
95
Nucleotide balance is tightly controlled by?
nucleotide balance tightly controlled CTPS & IMPDH - highly regulated
96
CTPS and IMPDH forms what to be more active?
filaments
97
What are the steps for the synthesis of nucleoside tri-phosphate?
1. Base specific kinases (generate DNPs)
| 2. NDP kinase (generate NTPs)
98
What enzymes are inolved involved in base specific kinases?
- GMP kinase
- UMP/CMP kinase
- adenylate kinase
99
What is NDP kinase?
(Generate NTPs) Highly Active Enzyme Broad Specificity with respect to both the sugar and the base (works with both rNDPs and dNDPs)
100
What is the one purpose of dNTPS?
DNA synthesis and repair Critical to cell viability
101
What are the major differences between rNTPS and dNTP?
The pyrimidine Thymine is only found as a dNTP The pyrimidine Uracil is only found as an rNTP (dUTP is very rapidly converted to dTTP) The 2’-hydroxyl group
102
How are deoxy Nucleotides made?
RNR and NDK turns rNDPs to dNTPs
103
What is the unique structure of Ribonucleotide reductase?
α2β2 tetramer
α-subunit:
-One Catalytic site - Two allosteric sites
β-subunits
-Generate a Tyrosine radical at β-site Electron travels to the α-active site ~35 Å away
104
What is at the center of the beta subunit of RNR?
Fe
105
how do the alpha and beta subunits of RNR interact?
The beta unit has a weak affinity for alpha unit until alpha is fully loaded. Then the tyrosine radical generated a the beta site transfers to the cysteine residue of the alpha active site. The transfer is assisted with the aromatic residues.
106
what are the two pathways that can be used to regenerate RNR?
Thio-redoxin (FAD/H2)
| gluta-redoxin
107
How does RNR balance the synthesis of dNTPs?
1. Specificity site:
what binds here controls binding of rNDP at the catalytic site
2. activity site:
what binds here controls the overall activity of the enzyme
3. dATP inhibits RNR active site at high levels
108
What happens when dATP binds to the active site of RNR?
Change in conformation that results inhibition. becomes α4β4 - octamer inactive complex. (ring)
109
What are the 5 enzymes needed for biosynthesis of dTTP from UMP?
1. ribonucleotidekinase(UMPkinase)
2. ribonucleotidereductase(RNR)
3. nucleotidediphosphatekinase(NDPKinase) 4. dUTPphosphatase(dUTPase)
5. thymidylatesynthase
110
Why can't dUTP be converted dUMP before becoming dTTP?
-dUTPase is a very active diphosphorylase.
-Keeps dUTP concentration low
Prevents incorporation of dUTP into DNA by DNA polymerase Energy is used to prevent errors!
111
What is the purpose of thymidylate synthase?
Biosynthesis of dTTP from UMP. Oxidation of THF accompanies carbon transfer.
112
How is THF regenerated for catalysis of dTTP?
Dihydrofolate reducatse uses NADPH. Serine then converts THF to N5,N10- methylene THF for use.
113
Nucleosides get degraded to what?
Uric Acid or β-Alanine and NH4+
114
How can nucleotides and nucleobases be recycled?
Salvage pathways using dietary, nucleic acid turnover, and DNA damage as sources.
115
What happens if a large amount of purines are degraded?
Production of uric acid causing high levels of uric acid which can lead to gout.
116
How can gout be treated?
Xanthine oxidase inhibitors.
117
In the degradation of purines, what must happen to adenosine before ribose-1-p can be removed?
must be deanimated to inosine.
118
What consists of Xanthine oxidase active site?
Fe-S clusters, FAD, Mo
119
What is alloxanthine?
-a mech based inhibitor of uric acid synthesis. Binds tightly to Xanthine oxidase. Increases levels of precursor, reducing gout.
120
What is Uloric?
Strongly binds to Mo of XO. Effective at low concentrations and will reduce gout.
121
What is Lesch Nyhan syndrome?
-Deficiency in Purine Salvage pathway.
-Complete loss of HGPRT activity is severe (X-linked recessive)
• Guanine and Hypoxanthine are consistently produced due to turnover of nucleic acids
• No salvage of guanine or hypoxanthine – Increased de novo synthesis of purines
• Excess production of uric acid – severe gouty arthritis
• Nervous system disorders - Motor disabilities, learning disabilities and behavioral
disorders
• Severe aggression and self-mutilation
122
What is Adenosine Deanimase (ADA) deficiency?
Impaired Purine Degradation and Salvage Severe Combined Immunodeficiency Syndrome (SCID)
B and T Lymphocytes can’t proliferate
• Can’t mount an immune response
• Patients are highly susceptible to infection
123
How can ADA deficiency be treated?
- Enzyme replacement therapy
- Bone marrow transplants
- Gene replacement therapy
124
What are inhibitors of Glutamine aminotransferases?
- Azaserine has antibiotic and anti-tumor properties
| - Acivicin is an anti-tumor agent
125
Why is Thymidylate synthase a target for chemotherapy?
-Inhibit Thymidylate Synthase - Block production of dTTP -
-Lethal to rapidly dividing cancer cells that need to
synthesize DNA
-5-fluorouracil and 5-fluorodeoxyuridine
126
5-fluoro-deoxyuridylate does what?
Suicide inhibits Thymidylate synthase.
127
What are anti-folates?
Analogs of THF that inhibit TS and DHFR
128
What are the antifolates?
* Methotrexate - Anti-neoplastic & immunosuppressant • Pemetrexed - Anti-neoplastic
* Proguanil - Anti-malarial
* Pyrimethamine - Anti-protozoal
* Trimethoprim - Broad-spectrum anti-microbial
129
What is methotrexate?
Substrate analog to DHFR, 1000 greater affinity than DHF.
- Low dose (rheumatoid arthritis)
- High (Cancer)
130
What is trimethoprim?
pyrimidine inhibitor of DHFR.
| binds 30000 better, inhibitor
131
Who is George Hitchings?
Discovered a lot of drugs (analogs) to attack nucleotide pathways.
132
Templated polymerization produces ..?
aperiodic polymers
133
What is the hybridization of DNA or RNA?
The pairing of two dNA or RNA to form non-covalent duplexes.
allows for template polymerization.
134
The backbone of duplex runs ?
antiparallel
135
What part of the ribose in a DNA duplex are connected by phosphodiester bonds?
3',5'
136
Phosphodiester back bones have a pka of?
1.0, they are negatively charged. Need counter ions to balance.
137
What are the factors that control the Tm of DNA and RNA hybridization?
* chain length
* solvent conditions (ionic strength, pH) • number of mismatched base pairs
* base composition (G:C to A:T ratio)
138
What does not provide energetic contribution to duplex formation?
H-bonds
139
What is the structure of DNA?
It is a right handed double helix, B-form.
140
What is the diameter of DNA?
20 angstroms
141
What is the rise per base pair of DNA?
3.4
142
How many base pairs per 360 degree helical turn?
10 bases
143
What is the angle per helical twist per base pair?
36 degrees
144
What is the length of the rise per helical turn?
34 angstroms
145
What is the structure of A DNA?
IT is more scrunched
146
When RNA forms a double helix, what conformation is it?
A conformation
147
What type of structure does Z DNA form?
Left handed double helix
148
What influences the Tm of DNA?
A:T to G:C base pairs. G:C pairs are better because of base stacking energy and the Vander Waals it produces.
149
Is the formation of the double helix driven by entropy nor enthalpy?
It is driven by entropy.
150
Contrast DNA and RNA polymerization, biological versus synthetic.
Biological=5-3
| synthetic= 3-5
151
Why did nature choose phosphorus?
- in biology they are reasonably good leaving groups.
- stable in water
- Intermediates are phosphoralyzed to keep it in cell compartments or to await another step
152
Why not use arsenic instead of phosphorus?
Phosphorous has a half life of 30,000,000 at 25 degrees in water while arsenic has 0.06s.
153
In human DNA, how many times does the phosphodiester backbone break each day?
on per 30000000 y
154
Which is more stable, RNA or DNA?
DNA. RNA breaks about 100 times more often than DNA.
155
What species has notably long DNA?
amphibians
156
Histones tend to be made of that are..?
positively charged to interact with negative backbone of DNA
157
Histone are structurally...?
octamers
158
What is the Metaphase chromatin organization?
It is when DNA wraps around a histones becoming nucleosomes and then the chromosome supercoils some more to allow more compaction.
159
DNA chromosomes are anchored to...?
chromosomal scaffolds.
160
In DNA, a twist is...?
The number of turns in DNA coiling, 10 bases per turn.
161
What is writhing in DNA?
It is how many times the DNA goes over the extra axis.
162
What happens when you increase twists in DNA?
you decrease the writhe
163
What is topological equivalence of DNA?
DNA wants 10 bases per turn, will create another head and adopt a negative super coil.
164
What does spooling of the DNA onto histones do?
it removes the negative twist, it increase the writhe.
165
What happens with the removal of histones in DNA topology?
makes twist more negative, facilitating local melting
166
What do topoisomerases do?
They are enzymes that modifying linking numbers (L).
| Must cleave one or both backbones. Must allow cleaved intermediates to diffuse away. Must re-ligate broken backbones..
167
What does Topsoimerase was 1A?
- Relaxes negatively supercoiled DNA
- can interlink (catenate) circle ssDNA
- covalent intermediate: Tyr on Top I transiently linked to the DNA
- Does not need ATP
168
What does topo II do?
passes one strand of dsDNA through another.
- Breaks and religates DNA on both strand
- symmetric dimers
- consumes ATP, releases ADP and Pi
- can interlink (catenate) two dsDNA circles
169
What is Top II mechanism?
G segment DNA comes in, Top needs ATP to allow it to hold onto the DNA, it lets go of the ATP and then the T- segment comes in and It needs more ATP to accept the T segment, this time to get rid of ATP needs to hydrolyze it out.
170
What is Gyrase?
It is bacterial Top II. It can coil without spooling, only enzyme that can known enzyme that can introduce negative supercoils.
171
Why are top II inhibitors important abx and chemotherapeutics?
These important abx are important for for inhibiting gyrase, important for killing bacteria but not eukaryotics. They are also important anti-cancer drugs.
172
Which abx specfically inhibit gyrase?
Ciproflaxin, Novobiocin
173
Which Top II inhibitors are anit-cancer drugs?
Etoposide, doxorubicin.
174
In dNA replication what does the nucleophilic attack?
The hydroxyl group does the nucleophilic attack.
175
Why is DNA replication semi-conservative?
Each of the daughter strands contain one of the original parent strands and one new strand.
176
Why are helicases like motors?
Hydrolyzes ATP to melt DNA and RNA . Does to change the linking number and uses a lot of energy.
177
Which phosphate does helicase helicase hydrolyze on ATP?
terminal (γ) phosphate on ATP
178
DnaB does what in the cell?
It is found in E. Coli and unwinds DNA for replication.
179
How many turns can helicase do per second?
100 turns, about 6000 rpms
180
What does DNA polymerase require to begin polymerization?
A primer, it can only add nucleotides to the free 3 OH' end.
181
What conformations does DNA polymerase have?
open and closed
182
What is in the active site of DNA polymerase when it is synthesizing DNA?
-Nascent strand, template strand, incoming NTP, Mg 2+ ions, with direction of polymerization 5-3. The leaving group is PPi
183
What are Dideoxy (dd) NTPs?
synthetic nucleotides that lack 3 OH ends, not normally found in nature, but essential for DNA sequencing.
184
What is the Sanger method?
You us ddNTPs and label them with 32P. They will combine and terminate nascent chains of template strands and then you use gel electrophoresis to determine the sequences to judge the sequence.
185
What did sanger win the nobel prize for?
- protein sequencing
| - DNA sequencing
186
What is sanger sequencing using dye termination?
same idea as normal sanger but instead of electrophoresis will count the concentration of the dyes to determine the sequence
187
What is Acyclovir (ACV)?
It is an important antiviral (pro) drug. Key drug against herpes. Activated by phosphorylation, a dGTP mimic. ACV-TP is the active form, lacks 3-OH, so is a terminator.
188
Why is ACV so effective against viruses?
It is more effective against viral TK and DNA than human TK and DNA.
189
What does each okazaki fragment require?
A new primer.
190
What enzyme makes new primers?
primase
191
What are nucleases?
phosphesterases that cleave DNA and RNA?
192
Why is Mg2+ important to DNA polymerase 1 active site?
It specifically bind to diphosphate leaving group, has a charge of 2+, and helps stabilize the structure so the leaving group can go.
193
How many polymerases do prokaryotes have, and what do they do?
pol I- erases primer and fills gaps on lagging strand
pol II- DNA repair
Pol III- Primary enzyme and DNA synthesis
194
How are prokaryotic okazaki fragments synthesized?
RNA primer laid down by primase, DNA pol III elongates them, DNA Pol I and ligase seals the gaps.
195
What are the general reactions for nuclease?
Phosphoester+ H2O --- acid + base
- M2+ cofactor
- some leave 3' - Pi while others leave 5' -Pi
196
What are exonucleases?
Nucleases that chew away free ends.
197
What are endonucleases?
Cut in the middle of a strand or duplex.
- some are non-specific
- some site specific
- some cut single, some cut double
198
What is make DNA pol I multifunctional?
in addition to its polymerase function, it has a 5'-3' exonuclease activity that removes RNA and DNA primers.
199
How does ligase seal nicks?
1. Ligase active site lysine forms covalent activated intermediate with NAD or AMP.
2. Adenosine-5 ́-diphosphate a ached to 5 ́ end of nick. Note 5 ́-5 ́ pyrophosphate linkage*
3. Nucleophilic attack
by 3 ́-OH seals nick, regenerates enzyme + AMP
200
What is Pol III holoenzyme?
an asymmetric dimer that catalyzes both leading and lagging strand synthesis.
201
What is SSB?
single stranded DNA binding protein that keeps melted DNA form re-annealing and protects exposed bases.
202
What is the sliding clamp in DNA synthesis?
-Keeps the polymerase complex form falling off, processivity factor .
203
In addition to being semi-cnonservative and discontinuous, DNA replication is also?
Bidirectional
204
Where does E. Coli begin replication on their chromosome?
Ori C, bind by set of proteins that melt DNA and place "replication factories" on the DNA, facing different directions.
205
How often can bacteria initiate DNA replication?
-more than once on a cell cycle. Each daughter cell will contain a chromosome that is already replicated.
206
How do bacteria terminate replication?
Top II decatenates chromosomes that are wound together.
207
How do eukaryotes solve the problem of replicating their chromosomes fast enough?
-Many origins of replication.
208
If you have hundreds of thousands of origins, how do you each one only once per cell division?
Coordinate to only turn each one on at once
Biochem 406
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