Unit 1 Flashcards
(158 cards)
1
Q
Gibbs Free Energy under non-standard conditions
A
ΔG=ΔG0’+RTln([products]/[reactants]
2
Q
Gas Constant
A
R is the gas constant:1.987 cal/mol ºK.
3
Q
Gibbs Free Energy related to equilibrium constant
A
ΔG0’ = -RT log K’eq cal/mol
or possibly ln
4
Q
Gibbs Free energy of redox reaction
A
ΔG’o = -nF ΔE’o
5
Q
Faraday Constant
A
96,500 joules/volt mol
6
Q
Difference between DNA and RNA nucleotides
A
DNA is missing a -OH on the 2’ ribose carbon
7
Q
Nucleotide
A
- nucleotide: base-sugar-phosphate
8
Q
Nucleoside
A
nucleoside: base-sugar (e.g.: deoxyadenosine)
9
Q
Base
A
A/T/C/G/U
10
Q
Solubility of Nucleotides
A
pyrimidines > purines; nucleotides > nucleosides > bases
11
Q
Gout and Lesch-Nyhan Disease
A
buildup of purines in our tissues. Purines are the least soluble = tissues that have these buildups have gross cellular defects
12
Q
AZT and DDI
A
Act as chain terminators (mimics nucleotide but doesn’t have 3’ OH. DDI (dideoxyinosine) can be used to inhibit HIV reverse transcriptase activity) AZT=Azidothymidine
13
Q
Avery, Cloud, and Mccarty
A
discovered that DNA is the important information carrier in our cells (live/dead virulent/nonvirulent experiments with mice)
14
Q
Chargaff’s Rule
A
Base pairing!
15
Q
Handedness of Helices in DNA
A
Right!
16
Q
Base pairs per turn of helix
A
10
17
Q
Nitrosamine
A
Converted to nitrous acid, a deaminating agent that causes mutations, found in cigarette smoke
18
Q
Puromycin function
A
an antibiotic that mimics the 3’ acceptor end of a tRNA that has an amino acid. It binds in the ribosome as it is translating & covalently attaches to a growing polypeptide chain –> terminates chain, prevents completion of translation.
19
Q
Cisplatin
A
Base alkylating agent (cancer)
20
Q
Actinomycin D and doxorubicin
A
a naturally occurring antibiotic that has also been used as a chemotherapy. “Intercalates”
into DNA (inserts a ring structure that can stack with DNA bases) and alters the double-helical structure.
Interferes with DNA replication and transcription
21
Q
Etoposide and Camptothecin
A
chemotherapeutics that target topoisomerases that relax DNA supercoiling
(super-twisting of the double-helix). Topoisomerases are necessary during DNA replication to avoid
supercoiling as the double helix is opened up to copy the strands. Topoisomerases must break the DNA
backbone to “relax” supercoiled DNA. Drugs that interfere with this process usually leave DNA breaks that
cannot be repaired
22
Q
Change-causing mutations
A
- Uncorrected replication errors
- Depurination
- Deamination
- Alkylation
- Pyrimidine Dimers
- Reactive Oxidative Species
- Base adducts
23
Q
Depurination
A
Purine (A/G) is removed, leaving only sugar phosphate backbone (unstable)
24
Q
Deamination
A
Amine group is removed from cC to form a U, which will then be base paired with A/T
25
Alkylation
Addition of a methyl group to the sixth oxygen on guanine--> O6-methylguanine
26
Pyrimidine Dimers
Thymine-thymine double bonds
27
Base adducts
BPDE addition to guanosine results in base matching with A by DNA pol
28
Direct Reversal of DNA Damage
1) repair of nick caused by DNA ligase in phosphodiester backbone
2) Removal of methyl from 6-O-methyl guanosine by MGMT
29
Mismatch Repair (MMR)
MutS and Mut L (prokaryotes) or MSH/MLH (Eukaryotes) scan strands and bind when they find an error. Then helicase unwinds, endonuclease cleaves, oligonucleotide is excised, polymerase refills, ligase seals.
New strand is ID'd in Eukaryotes by increased number of nicks.
30
Base Excision Repair (BER)
*For mutations that are do not cause structural changes in DNA and are not targeted by Nucleotide excision Repair (ex. C--> U or methylation)
Depends on glycosylase, which cuts the wrong base away from the phosphosugar backbone. The abasic site is then removed by 2 endonucleases (3' and 5' cuts), refilled by DNA Pol, and sealed by ligase.
31
Nucleotide Excision Repair (NER)
*Corrects issues that distort DNA structure and block polymerase function (ex. thymine dimers and BPDE base adducts)
Distorted DNA is recognized by protein complex that binds. Helicase unwinds (part of TFIIH complex) and endonucleases cut either sides of ~30 bp oligonucleotide. Pol fills in gap, ligase sease.
32
Diseases caused by mutation in NER genes
xeroderma pigmentosum, cockayne syndrome, tricothiodystrophy
33
Repair pathway for big mutations that cannot be fixed by NER
Trans-lesion synthesis
34
Protein Kinases important for DNA damage checkpoint
ATM and ATR
35
Mismatch repair defect associated disease
hereditary non-polyposis colorectal cancer
36
DNA Polymerase I
helps correct & repair DNA. Replaces the RNA primer with DNA: has 5’-3’ exonuclease activity to remove RNA, then 5’-3’ polymerase activity to fill in the gap.
37
DNA Polymerase III
MAJOR REPLICATIVE ENZYME – has a sliding clamp that keeps it attached to DNA over long distances
38
RNA Polymerase I
Makes ribosomal RNA= is the busiest of the all the pols – makes over 90% of the RNA in our cells
39
RNA Pol II
mRNA, snRNA, miRNA, lncRNA
- has unique C-terminal domain (CTD) on large subunit
o This binds to proteins that help regulate elongation & processing of the mRNA transcript
40
RNA Pol III
tRNA
41
E Coli Polymerase
1 type of polymerase for everything
42
alpha-amantin
a toxin found in mushrooms. Binds to RNA pol II at the site of the bridge helix. Prevents translocation, elongation of the growing mRNA chain --> kills you
43
rifampicin
Antibiotic: binds bacterial RNA polymerase and blocks the RNA exit channel
44
Components of RNA Pol II initiation complex
TFIIA, TFIIB, TFIID (TATA-binding protein), TFIIE (also has helicase activity and ATPase), TFIIH(also participates in NER, acts as helicase.
CDK7 (Part of TFIIH) phosphorylates CTD on RNA Pol II during promoter clearance
45
TATA binding protein function
TBP binds in minor groove and directs assembly of initiation complex
46
5' Capping Steps
1) remove triphosphate on 5' end with triphosphatase
2) Transfer GTP to the chain backwards (5'-5') with guanylyltransferase
3) Methylate GTP with guanylyl-7-methyl transferase
47
Purposes of 5' cap
1) removes 5' triphosphate
2) makes 5' end resistant to exonucleases
3) Helps with splicing and processing with cap-binding complex
4) Translation factor eIF4E recognizes cap and transports mRNA to ribosomes
5) degradation of 5' cap is a sign for mRNA degradation
48
Conserved sequences in introns
5' GU........A...............AG 3'
49
Genetic Disorders caused by Splicing
Marfan's syndrome - mutations in fibrillin transcript. They are tall and prone to aneurysms
Abnormal splicing of CD44 gene promotes tumor metastasis
50
Binding sites of snRNA's in splicing
1) u1snRNA==>5' GU
2) u2 snRNA ==> Branch point A
3) u2AF snRNA ==> 3' AG
51
Termination Codons
UAG, UGA, UAA
52
poly A site consensus sequence
AAUAAA
53
Poly-A tail formation
1) new mRNA is cleaved at the Poly-A consensus sequence
| 2) Poly-A tail is added and capped with PAB (poly-A-binding protein)
54
Poly-A tail functions
1) Protection from degradation
2) export from nucleus
* cancer cells often have shorter polyA tails to avoid detection
55
Overexpression of EIF4E
Leads to malignant transformation
56
Treatment of Spinal Muscular Atrophy
Rescue of SMN1 mutations by altering splice sites
57
alpha-Thallasemia
Several types of anemias. Can be caused by mutation of AAUAAA or mutation of gene in beta-gobin promoter
-Mutations also often disrupt alpha helix structure
58
Hemophilia-B-Leyden
X-linked disorder that involves clotting
Mutation in Promoter IX gene
Androgen receptor can bind nearby and promote translation, so before puberty they only make 1% of factor IX and after puberty they make 60% of normal factor IX
59
Fragile X syndrome
CGG repeats in the FMR1 gene, leading to increased methylation of the cytosine (CpG) and increased gene silencing
60
Targets for drugs attacking DNA metabolism
- Synthesis of precursors (dNTP)
- Intercalation (getting in the middle)
- Covalently binding bps
- Topoisomerases
61
Craniosynostosis
Defect of homeodomain protein that binds too tightly to MSX2 gene, leading to premature closure of skull
Gain of fxn
62
Androgen Insensitivity Syndrome
Mutation of DNA-binding region or ligand (androgen) domain of androgen binding protein, a zinc finger DNA binding protein. Secondary sex characteristics are not developed and person may be infertile
63
Waardenburg Syndrome Type II
Mutation in a gene for a transcription factor that plays a role in development of melanocytes. Characterized by deafness and pigmentation anomolies
64
TF's that form heterodimers (combinatorial control)
zinc finger, bZIP, bHLH
65
Possible modifications of histone lysine residues
acetylation, ubiquination, phosphorylation, methylation
66
Rubinstein-Taybi Syndrome
Mutation in gene coding for CBP protein (a HAT)
| results in mental retardation, craniofacial abnormalities,
67
Leukemia
Gain of function fusion proteins --> sometimes transcriptional factors will fuse with HDAC or HAT, altering the activity of the regulators
68
Tamoxifen
Breast cancer drug that works by binding to estrogen receptor and recruiting co-repressors
69
Examples of nuclear entry protein regulation
NF-KappaB is sequestered in cytoplasm by IKappaB
| Ca2+/Calcineurin regulate entry of NF-AT into nucleus (immune response and heart effects)
70
Examples of regulation of amount of activator/repressor
Levels of beta-catenin in a cell is regulated by APC, which is often mutated in familial adenomatous polyposis (colon cancer
MDM2 binds p53 protein and leads to its degradation. (p53 is a VERY important tumor supressor. "guardian of the genome"
71
Example of inhibition of DNA binding activity of a sequence specific binding protein
Id proteins bind to HLH proteins as a heterodimer, but prevent binding to DNA because they lack a DNA binding domain
72
Example of protein modification that alters activity of a sequence specific binding protein
CREB (phosphorylated by G protein activates a HAT (CBP) which recruits RNA Pol II
73
High Energy Bonds
ATP is an example of a phosphoanhydride bond.
Acetyl coA contains high energy thioester bonds (C-S)
There are 3 types of high energy phosphate bonds:
1. Phosphoanhydride (ATP)
2. Phosphocreatine (P-N)
3. Phosphoenolpyruate bonds (C-O-P)
74
Rapamycin
dephosphorylates 4eBP, which then goes to bind and inactivate eIF4E
75
function of eIF4E
Initiates eukaryotic translation. Binds 5' cap, recruits other IF's and small subunit. Small subunit then scans down until it finds AUG
76
Bacterial Initiation
EF1 and EF3 bind small subunit, which binds the shine delgarno sequence, placing the AUG in the P site
eF2 brings over a special formylmethionine. Then GTP hydrolysis causes release of IF's and binding of the large subunit
77
Polycistronic bacterial genes
Bacterial genes may contain many AUG sites, and any with a Shine Delgarno sequence will be translated. These genes often exist in operons.
78
Factors that drive regulation of Translation
Varying the activities of elongation or initiation factors
mRNA structure and sequence
Binding of proteins to the mRNA
Action of small molecules (such as antibiotics)
79
mTOR
A central player in many processes, regulates translation
| ex. Induces phosphorylation of e1F4E-binding proteins
80
Regulation of eIF4e
eIF4e is inhibited by eIF4e-bp. eIF4e is turned off when it's phosphorylated (via mTOR)
Stress and Rapamycin--> dephosphorylation of 4eBP
81
Regulation of eIF2alpha
Phosphorylation inhibits it, which prevents binding of initiator tRNA from being delivered to the ribosome
Phosphorylated by: Interferon (produced when cell is infected by virus), other cell stressors
82
IRES-driven translation
*In some cases, viruses cleave eIF4G, shutting down cap dependent initiation and only allowing IRES-driven translation
83
a) Hemoglobin wayne mutation
| b) Hemoglobin constant spring mutation
1) 3' frameshift mutation
| 2) Sense mutation
84
mRNA editing example
apoB is edited by cytosine deamination (C--> U), creating a stop codon and a truncated protein in the intestine, so the protein is differentially expressed in the liver and intestines
85
Kozak Sequence
RNA sequence upstream of an AUG that modulates the likelihood that the small subunit will bind on a given AUG site
86
Antibiotics that target the ribosome
Streptomycin, Erythromycin, Tetracycline, Chloramphenicol
87
Translational regulation of intercellular Iron levels
1. Transferrin binds iron extracellularly
2. TR (transferrin receptor) transports transferrin-Fe compound into cell
3. Ferritin – sequesters excess Fe
4. Iron Response Element (IRE) – sequence in transferrin receptor mRNA that binds to IRE-BP
5. Iron Response Binding Protein (IRE-BP) – bind Fe & regulate expression of ferritin & TFR
Basically = during times of low iron, IRE-BP is free to bind to IRE on mRNA that encodes for transferrin receptor; prevents degradation of mRNA & translation occurs. More Fe gets into cell. During high iron, IRE-BP is bound to iron, and mRNA gets degraded.
88
Amino Acid H-bond donor mnemonic
THYNQ WRKS (Thymine, histidine, tyrosine, asparagine, glutamine, tryptophan, arginine, lysine, serine)
89
Amino Acid H-bond acceptor mnemonic
Y STD? GQ : Tyrosine, serine, threonine, aspartate, glycine, glutamine
90
Essential Amino acid mnemonic
WHR FVT MILK (Tryptophan, histidine, arginine, phenylalanine, valine, threonine, methionine, isoleucine leucine, lysine)
91
Hydroxyproline
-Important in collagen formation
-Vitamin C is necessary for enzyme to work
Disease failure--> scurvy
92
Gamma-Carboxy Glutamate
-Occurs in clotting factor proteins
-Enzyme requires vitamin K (Vitamin K deficiency causes clotting disorders)
Warfarin is a blood thinner that inhibits this enzyme
93
Glycosylation
O-linked: Serine/Threonine
N-Linked: Asparagine
O-linked is basis of ABO blood typing
Disorders of gylcosylation lead to congenital disorder of glycosylation -->hypotonia, psychomotor retardation, seizures
94
Acetylation and methylation
Lysine and Arginine make histones extremely positive and are targets for acetylation and methylation
-this leads to histone modification and is a target for cancer drugs (ex. vorinostat targets a HDAC)
95
Reversible phosphorylation
-Ser/Thr/Tyr
-catalyzed by kinase/phosphatase and useful in signal transduction
Ex. Gleevec is a leukemia treatment
In lukemia, bcr-abl is a mutated kinase that is permanently on, Gleevec targets the kinase domain and turns off oncogenic effects
96
Ubiquination
- protein marked with other protein, multi-ubiquitin chain marks protein for degradation
- in multiple myeloma, proteasomes are overactive. Ubiquitination of proteasomes can inhibit cancer activity
97
sickle cell anemia mutation
missense mutation involving one amino acid change (glutamate-->valine)
(**Note, there are also normal healthy differences in people that don't affect function called polymorphisms)
98
Proteases
digest peptide bonds of any protein (ex. Trypsin in digestion)
* many proteins are activated by cleavage (insulin, trypsin, some transcription factors, blood clotting cascade involves protein cleavage)
99
Peptidases/ specific proteases
HIV protease-cleaves a specific protein that is needed for HIV to reproduce (inhibited by crixivan)
Angiotensin II formed by cleavage by ACE (inhibited by captopril)
100
Alpha helix
Carbonyl-O binds with i+4 N-H group (both in backbone)
usually right handed
side chains flare out to the outside
Residues 1 and 8 stack
- Each turn is 3.6 residues and 5.4 Angstroms
Fits well into major groove of DNA
101
Leucine Zipper Domain
2 alpha helices with 7 residue turns, and a Leucine or isoleucine every 7 positions
102
Helix stabilizers and breakers
Breakers: Proline and glycine
Stabilizers: Alanine and Leucine
Mostly alpha helixes: Structural fibrous proteins (keratin, myosin) and globular proteins (hemoglobin/myoglobin)
103
Beta Sheets
ex. fibroin silk, spider webs, immunoglobulin, pepsin, HIV protease
- Also H-bond between 1 and 4 residue
- 180 turn spans 4 acids and usually has a glycine in position 3 or a proline in 2 (~6% of prolines are cis)
104
Amino Acids that promote beta sheets
Trp, Ile, Val
105
Amino acids common in beta turns
Gly and Pro
106
Circular dichroism analysis
Measures absorption difference of L and R circularly polarized light. the curve depends on the secondary structure (chain conformation)
107
Main classes of tertiary proteins
Fibrous: typically insoluble, made of a single secondary structure
Globular: typically water soluble, or lipid soluble (membrane proteins)
108
Dissociation constant for protein binding (Kd)
** Opposite of equilibrium
kd=[P][L]/[PL]
Kd==> when 50% of ligand is bound to a protein
109
Logic behind embedding heme group in myoglobin/hemoglobin
```
Myoglobin needs to bind oxygen to deliver to tissues, but protein chains have no affinity for oxygen.
Some transition metals can bind oxygen, but if just floating in fluid, they would generate free radicals.
Free heme (an organometallic compound) can bind oxygen, but free heme would be oxidized from Fe2+ to Fe3+ and would no longer be able to bind oxygen.
SO --> heme + 4 globular protein subunits = hemoglobin
heme+1 globular protein subunit= myoglobin
```
110
Binding of Heme to myoglobin
Iron in Heme is coordinated with a Histidine residue from the myoglobin protein
111
Mxn of CO poisoning
CO can also bind the O2 binding site in Heme and it binds WAY better than O2
-A protein pocket decreases the affinity for CO, but it still binds much more strongly
112
Why is Myoglobin not a good O2 transporter?
Myoglobin has great affinity for oxygen in lungs, but the affinity is too high in tissues --> would not release the oxygen.
113
Why is hemoglobin a good O2 transporter
Sigmoid binding curve due to positive cooperativity (first binding increases binding affinity at other sites)
-4 hemoglobin subunits (2 alpha, 2beta) all interact
Oxygen binding triggers a switch from T (tense, lower O2 affinity) to R (relaxed, higher O2 affinity) state.
114
Bohr Effect
blood in lung has higher pH than blood in metabolic tissues. O2 binds well in higher pH conditions and releases well in lower pH conditions. Thus, the pH conditions of the various tissues encourage binding/release of O2 --> increases transfer efficiency
115
Methods of protein denaturation
Heat, pH, chemicals (urea, guanidium (dissolves polar), detergent, organic solvent)
116
Ribonuclease refolding experiment
Proteins, even after being denatured, can refold & regain activity completely --> thus, the folding of a protein relies only on the primary structure. The environment provided by the cell is not always required.
When you denature, you disrupt tertiary and quaternary structures
117
Hsp70/Hsp40
Protein folding chaperones
- induced at higher temperatures
- bind to hydrophobic regions of proteins & prevent them from aggregating
- can help with transport (unfolded) across cell membranes
118
Chaperonin
- GroEl/GroES complex in E. coli
- Form a complex together with ATP binding that encourages folding of a protein (alternatively hydrophobic/hydrophilic environments)
- 7 subunit rings
119
Protein disulfide isomerase
- Helps with protein folding
| - gets rid of improper disulfide bonds and reforms them correctly
120
Peptide Prolyl isomerase
Turns Proline from trans--> cis for incorporation in beta turns
* cyclophilin is a PPI that activates calcineurin (stimulates immune response)
cyclophilin is inhibited by cyclosporin (immunosupressant)
121
CF protein misfolding
defects in cystic fibrosis transmembrane conductance regulator (CFTR)
most common mutation is the deletion of F508
F508 deletion causes protein misfolding
122
Prion disease misfolding
```
prion protein (PrP) changes from alpha-helices to mostly beta-pleated sheets --> insoluble, aggregate --> become deadly PrpC-healthy
PrpSc--> prion disease
```
123
Alzheimer's misfolding
plaque made up of the b-amyloid (Ab42) and the tangle made up of cytoskeletal protein tau
124
Parkinson's misfolding
Lewy bodies (protein accumulation)
125
amyloidosis
accumulation in areas other than the brain. Includes heart, lung, kidney
126
Gel filtration chromatography
separates by size; small proteins go slower, getting stuck in little bead traps
127
Ion exchange chromatography
separates by charge. Cation exchange resin is negatively charged --> binds cations. Negatively charged proteins go faster
128
Affinity Chromatography
resins have immobilized ligands; separates based on affinity
129
SDS Page (polyacrylimide gel)
Gel electrophoresis bind with SDS, so they have uniform size. Proteins migrate through polyacrylamide gel and the smaller ones move faster
130
Mass Spec
Determines mass of a protein
131
Edman degradation
determines the sequence of a protein, one aa at a time, from the N-terminus end
132
Western blot
immunological studies of proteins. Take a protein, run it through gel. Blot it onto a PVDF membrane. Treat with an antibody, wash, then detect if the antibody bound through another molecule that will bind to the antibody, typically with something attached that you can visualize.
133
RNA interference
An endogenous gene silencing mechanism, present in virtually all
eukaryotic cells, by which short double-stranded RNA molecules induce translational inhibition
and/or degradation of mRNAs containing partially complementary sequences.
134
Gene knockdown
An experimental technique used to reduce gene expression using sequencespecific
oligonucleotides, typically by RNA interference (RNAi) or antisense mechanisms.
135
RNA-induced silencing complex
The catalytic effector complex of RNA interference
(RNAi)-mediated gene silencing. The RISC is a multiprotein complex that incorporates one
strand of a small interfering RNA (siRNA) or microRNA.
136
siRNA
endogenous siRNA's act as transcriptional repressors (heterochromatin formation), mRNA clevage, mobile element silencing
137
Argonaute
** RISC component
A family of proteins that bind to small RNAs and that are conserved in all
domains of life. They mediate target recognition via base‐pairing interactions between their
bound small RNA and complementary coding or non‐coding RNAs.
-Has endonuclease activity in miRNA decay of mRNA (but not all have slicer activity)
138
pi RNAs
Mobile element (transposon) silencing and regulation of transcription
Small RNAs that are associated with the PIWI protein
complex and that emanated from transposon-like elements
-Primarily related to germ-line regulation and gametogenesis
139
Guide strand of mature siRNA or miRNA
```
In mature, duplex small interfering RNAs (siRNAs) and microRNAs (miRNAs),
the strand with intrinsic sequence characteristics that favour its association with the RNAinduced
silencing complex (RISC). It is usually turned into the active siRNA or miRNA.
```
140
Passenger strand of mature siRNA or miRNA
: In mature, duplex small interfering RNAs (siRNAs) and microRNAs
(miRNAs), the strand with less preference for RNA-induced silencing complex (RISC) loading. It
is usually quickly degraded or can turn into a miRNA
141
Aptamers
Oligonucleotides (DNA or RNA) selected to bind with high affinity to defined
structures.
142
Enhancer RNA (eRNA)
a class of lnc RNA that is implicated in enhancer function
143
RNA-mediated Transcriptional Gene Silencing (TGS)
Typically uses repressive chromatin remodeling
144
RNA-directed DNA Methylation (RdDM)
a plant TGS pathway characterized by the
| involvement of two specialized RNA polymerases, Pol IV and Pol V.
145
miRNA
~21 nt's
pretty specifically act through translational repression of mRNAs and mRNA degradation. There are also limited cases of translational activation.
Perfect match--> degradation
Imperfect match-->repression
146
miRNA targeting
- miRNA binds with 3'UTR of mRNA and guides effector complex to target
- can also target 5' UTR or coding region- binding region affects function
- miRNA's can be sequestered by lnc RNA's and crcular RNA's
147
Dicer
– The ribonuclease of the RNase III family that cleaves miRNA precursor
(pre-miRNA) and double-stranded RNA molecules into 21–25-nucleotidelong
double-stranded RNA with a two-base overhang on the 3’-ends
148
Restriction point in cell cycle
Point in G1 where the cell decides whether it will replicate based on cell size, hormones, etc.
149
Main goal of somatic cell cycle (besides creating another cell)
is to ensure exact duplication of the genome in S phase followed by exact of division of the genome in M phase to produce identical daughter cells.
150
Fluctuating CDK activities
* M- high CDK prevents building Pre-Replication Complex
* G1- low CDK allows building of Pre-RC
* S- high CDK activates replication and prevents any more Pre-RC building
151
Retinablastoma Protein
Rb (Retinoblastoma protein) is an inhibitor of
the cell cycle. When cells divide, the Rb inhibition must be removed. Rb is inhibited by CDK phosphorylation resulting in an “inhibitor of an inhibitor”, that is a double negative, which is a positive: Rb - X CDK - = +. Rb is known tumor
suppressor in that loss of Rb results in tumors in retinoblastoma and also in other cancers (e.g. small cell lung cancer).
152
Two families of CDK inhibitors
Cip/kip and ink4 -- all are encoded by CDKN gene.
Cip/kip are non-specific
ink4 is specific to certain CDK's
*one ink4 enzyme is a tumor suressor
153
MCM DNA
an important protein in the pre-rc
154
Pre-RC activation proteins
a S-CDK and DDK result in MCM DNA activation and loading of other initiation complex proteins
155
DNA Replication and Damage Checkpoint proteins
p53 and Chk2 inhibit CDK cyclin2 complexes
Li-fraumeni syndrome--> mutation in p53 or Chk2 leads to high susceptibility to cancer
ATM and ATR are also important kinases
-mutations are like li fraumeni but with neurological symptoms
156
BRCA1/2
BRCA1/2 regulate the ATM/ATR phosphate pathway. Mutations lead to high likelihood of breast or ovarian cancer
BRCA1 and 53BP1 are pivotal regulators directing cell towards HR or NHEJ, respectively, and BRCA1 is pivotal for proper HR function
157
5 sources of DS DNA breaks
1. Endogenous (immune system rearrangements [critical for Igs, T Cells], single strand breaks during DNA replication, meiosis)
a. VDJ recombination – needed for Ig rearrangements
b. DNA replication
c. Successful meiosis
2. Exogenous
a. Ionizing radiation [cosmic rays and soils]
b. Medical imaging and treatments
158
Major considerations in ID-ing next-gen sequencing variant
1) Read depth/coverage
2) Error Rate
3) contiguity- short read sequences won't associate haplotypes, long reads will.
