B1W2: Mtc (Except Nucleotide Metabolism)

Front 1

G1

Back 1

Cell either senescence/differentiation (Go), apoptosis or proliferation (cell cycle)

Cyclin D up, stable throughout

P27 down, CDK 4 used

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Checkpoint 1

Back 2

Regulated by Cyclin D/CDK4

1. growth factor --> myc --> cyclin D

2. cyclin D binds to CDK4

3. cyclin D/CDK 4 phosphorylates Rb

4. P-Rb releases E2F

5. E2F activates genes for cyclin E and cyclin A

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Checkpoint 2:

Back 3

G1/S "Restriction" checkpoint; cyclin E/CDK-2

1. Cyclin E to CDK 2

2. Cyclin E/CDK 2 phosphorylates target proteins

AT SAME TIME

1. p21 induced by p53

2. Binds to cyclin E/CDK-2 (if wants to inhibit)

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Synthesis:

Back 4

Synthesis of chromatin; histone proteins; ~ 4 years

--cycli E and A rise; CDK 2 used

Front 5

Cyclin-Dependent Protein Kinases

Back 5

Attach PO4- to S, T, Y

Cyclin regulatory unit

CDK catalytic

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Checkpoint 3

Back 6

Thru S phase; regulated by cyclin A/CDK 2

1. Cyclin A binds to CDK 2

2. Cyclin A/CDK 2 phosphorlates proteins in DNA replication complexes

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G2

Back 7

Preparing for M phase; 1 hr, centrosome duplicates

Increase in Cyclin B, use CDK 1

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Checkpoint 4

Back 8

Dephosphorylation cyclin B/CDK 1; G2 --> M phase

1. Cdc25 phosphatase DE-PHOSPHORYLATES CDK1/Cyclin B to activate

2. Cyclin B/CDK causes phosphorylations in nucleus that cause:

--nuclear envelope breakdown

--assembly miotic spindle

--metaphase arrest

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M

Back 9

mitosis, 1 hr

Front 10

External proteins reg. cell cycle

Back 10

Hormones, cytokines, growth factors

Activators: FGFs, IGFs, Wnts

Inhibitors=TGFB

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Internal proteins regulating cell cycle

Back 11

--early response=myc, fos, jun

--delayed response= cyclins + CDKs

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5 categories of cancer

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1. Proto-oncogenes

2. Tumor suppressor genes

3. Genes that regulate apoptosis

4. Telomerase

5. Genes that repair DNA

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Proto-onco genes

Back 13

Become oncogenes when mutated, forming tumors and over activation of proteins

--Cell membrane receptors GF (EGFR=lung carcinoma, Src=sarcoma/colon cancer)

--Cyclins/Cdks

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Tumor Suppressor Genes

Back 14

Inactivated in tumors

p21: inhibits CDK4 and CDK 2

p53: induces apoptosis, gene encoding p21 (cigarette smoke mutates; 3 nucleotides added)

Rb: binds and inhibits E2F-1

BRCA-1 and BRCA-2: repair DNA

Front 15

Genes regulate apoptosis

Back 15

Mutations overly inactivate apoptosis

Apoptosis: macrophages secrete TNF --> TNF receptor --> balances pro/anti apotic factors (pro-apoptotic factors) --> cytochrome C leaks --> caspase --> chromosome frag. --> cell disruption

Bcl-2 Gene: causes B cell lymphoma; aptosis overly inhibited

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Telomerase

Back 16

When mutated in somatic cels, activates, cells become immortal

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Genes that repair DNA

Back 17

i.e. BRCA, mutations pile up, cell cycle won't stop, colo-rectal cancer

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Current cancer remedies

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Clinical testing for BRCA mutations, bladder cancer antigens, prostate antigens; non specific treatment

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Future Remedies

Back 19

Specific treatments that

1. Target metastasis (drugs inhibit metallo-proteases)

2. Target angiogenesis (prevent BV growth)

3. Target specific molecules

--inhibit tumor cells (rituxan to CD20 in B cell lymphoma, herceptin in breast cancer)

--activate mutated proteins (p53 or replace with Advexin)

--destroy pathogenic RNAs (siRNA therapy)

Front 20

Properties DNA Synthesis

Back 20

Semiconservative (Meelson-Stahl)

5' --> 3' synthesis (creating leading/lagging strand)

Template and 3' OH primer needed (does nucleophillic attack on alpha phosphate of incoming triphosphate, hydrolyzation providing energy)

Semi discontinuous: lagging strand has Okazaki fragments placed in direction replication forks, DNA polymerase 5' to 3'

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Steps DNA Replication

Back 21

1. Separation strands (in multiple places in eurkaryotes); SSB

2. Formation replication fork (primase, helicase like DNA G, DNA B protein)

3. Chain elongation

--> DNA Polymerase III on both strands, DNA polymerase 1 on lagging strand (excise primer)

4. Removal of primers

--> DNA ligase seals nick, gyrase coils up (DNA topoisomerase III)

Front 22

DNA Polymerase I Properties

Back 22

1. 5' to 3' polymerase: make new DNA

2. 5' to 3' exonuclease: remove primers lagging strand

3. 3' to 5' exonuclease: removes bond base pairs (proofread)

a. mispairs

b. confirmational change to 3' -- 5' exonuclease site

c. hydrolyzes mismatch

d. 3' terminus positioned into polymerase site again

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Processiveness

Back 23

How long polymerase can synthesize DNA without letting go

DNA polymerase III move processive because has more subunits

DNA polymerase I low processivity because how to exonuclease activity

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Telomere

Back 24

Has repeating GGGGTT hexonucleotide sequence because a little bit is lost from lagging strand at 5' end every replication (where first primer was; DNA polymerase I cannot fill in)

Tells lifespan of cell

Telomerase repairs in germ line cells; shouldn't be active in somatic cells; has CA repeats and extends 3' end using its own RNA as a primer

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4 Types of DNA Damage

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1. Mis-incorporation of nucleotides during DNA replication

2. Inherent nonenzymatic chemical instability of bases

3. Environmental mutagens

4. Ionizing UV light

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Mis-incorportion of Nucleotides during DNA replication

Back 26

DNA bases can be in rare tautomer forms; would change bae pairing (i.e. A w/ C)

Front 27

Inherent nonenzymatic chemical instability of bases

Back 27

Cytosine can be deaminated naturally to become uracil

N-glycosidic bond can be hydrolyzed in purines

Front 28

Environmental mutagens

Back 28

Chemicals=cause deamination (C to U, G to X) via nitrous acid precursors (NaNO2, NaNO3, nitrosamine)

Alkylating agent of N-7-guanine=nitrogen mustards, oflatoxin, benzoapyrene, AAF (N-2-acetyl-2-aminofluorene)

Front 29

Ionizing UV Light

Back 29

Causes pyrimidine dimers; light makes planar bases stack

Intercalating agents

Front 30

5 DNA Repair Systems

Back 30

1. DNA methylation/mismatch repair

2. Base-excision repair (C becomes U)

3. BASE Excision Repairs in Humans

4. Direct Repair

5. Recombination Repair -- for double stranded breaks, cross links and lesions

Front 31

DNA Methylation/Mismatch Repair

Back 31

--New strands after DNA replication = not methylated, old strand is

--MutH binds to mismatch, MutL and MutS complex move to find MutH

--MutH cleaves unmethylated strand, and it is degraded by exonuclease

--Gap filled in correctly with polymerase

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Base-excision repair (C becomes U)

Back 32

--DNA glycosylase recognizes damaged base and cleaves backbone

--AP endonuclease cleaves backbone

--DNA polymerase I removes base at 3' end and repairs

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Base-excision repair in humans

Back 33

Exonuclease binds to DNA at bulky group, cleaves on both sides

13 or 29 nucleotides removed with helicase

DNA polymerase fils in gaps

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Direct Repair

Back 34

i.e. removing alkyl grps at O6 of guanine with enzyme MGMT, directly removes (has S that binds to CH3)

try to inactivate alkylating enzyme in chemo

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Recombination Repair

Back 35

For double stranded breaks, cross links and lesions; need info from homologous chromosome to fix

Happens from radiation/ionizing/oxidation reactions

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Defects in DNA Damage Repair

Back 36

Leads to increase in sensitivty to DNA damaging agents, leading to cancer

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Xeroderma Pigmentosum

Back 37

Defect in nucleotide excision repair; thymine dimers

Sensitive to UV light, therefore get skin cancer

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Werner's syndrome

Back 38

premature aging because DNA helicase messed up

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Bloom Syndrome

Back 39

Small body, UV sensitivity, lesions, immunodeficiency, cancer, diabetes, lung disease because ligase I for DNA repair damaged

Similiar to Fanconi's anemia, Ataxia telognectasa and Gardner's syndrome

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RNA Synthesis: Eukaryotes

Back 40

--Post translational modifications

--three diff. RNA polymerases

--mitochondria have own polymerases

--monocistronic (carries info to make only one protein per mRNA chain)

--contains microRNA

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RNA Synthesis: Prokaryotes

Back 41

--transcription/translation take place in same compartment

--only one RNA polymerase

--Polycistronic

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Types of RNA (4)

Back 42

mRNA: encode the amino acid sequences of all polypeptides found in cell

tRNA: match specific amino acids to triplet codons in mRNA

rRNA: catalytic components to make amino acids

microRNA: regulate expression of genes via binding to nucleotide sequences

Front 43

Similarities of Transcription Steps to Replication Steps

Back 43

3'OH attack when adding bases

Polymerases still used

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Three steps of transcription

Back 44

Initiation, elongation, termination

Front 45

Initiation:

Back 45

RNA polymerase with sigma factor binds to DNA promoter

--diff. sigma factors recognize different promoters

--as you increase the specificity of the sigma factor, you decrease affinity

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Elongation

Back 46

5'-3' direction

Sigma factor disassociates once it begins and a transcription bubble forms

Chain hangs loosely because of positive and neg. supercoils, which must be relieved by a topisomerase

Front 47

Protein Independent Termination

Back 47

1. RNA polymerase hits GC rich area followed by AT

2. Polymerase pauses at this; hairpin starts forming

3. Affinity of polymerase to strands less than sRNA, so hairpin forms and causes dissassociation

Front 48

Protein Dependent Termination

Back 48

p (rho) protein helicase binds to rut site; uses ATP to migrate along the strand, separating it from the polymerase

Front 49

Four Diff. Types RNA Polymerases

Back 49

Prokaryotic RNA polymerase

Eukaryotic RNA polymerase I

Eukaryotic RNA polymerase II

Eukaryotic RNA polymerase III

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Prokaryotic RNA polymerase

Back 50

Only one, multiunit enzyme (including holoenzyme with sigma factor)

Lacks 3'-5' exonuclease activity but it doesn't matter because RNA is not the heritable molecule; can have all the mistakes it wants

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Eukaryotic RNA polymerase I

Back 51

synthesizes pre-rRNA

Front 52

Eukaryotic RNA polymerase II

Back 52

synthesizes mRNA

Front 53

Eukaryotic RNA polymerase III

Back 53

makes tRNAs

Front 54

RNA Transcription Inhibitors--all

Back 54

Rifampicin

Alpha amanitin

Actinomycin D

Daunorubicin

Cordyepin

Front 55

Rifampicin

Back 55

Inhibits bacterial RNA polymerases

Used in Tb cures

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Alpha amanitin

Back 56

Inhibits polymerase II

Mushroom toxin

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Actinomycin D

Back 57

Intercalates between GC base pairs of DNA

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Daunorubicin

Back 58

Intercalates between base pairs

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Cordyepin

Back 59

Chain terminator of RNA that lacks 3' OH

Front 60

CFTR Channel

Back 60

Chloride channel that is mutated by splicing issue; causes cystic fibrosis

Front 61

Post-Translational Modifications of mRNA (3)

Back 61

Addition of 5' Cap

Addition of Poly A Tail

Splicing

Front 62

Addition of 5' Cap

Back 62

Enhances stability (from nucleases), efficiency

--7 methyl guanosine added by guanylytransferase

--methyl groups sometimes added to riboses by methyltransferases using S-Adenosyl Methionine (SAM) as donor

Front 63

Addition of Poly-A Tail

Back 63

Enhances stability, aids in translation

--added polyadenylation signal in 3' UTR

--enzymes use the signal to regulate RNA translation (?)

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Splicing without snRNAs

Back 64

5' donor site and 3' acceptor site

1. 2' OH attacks 5' splice site

2. New OH attacks 3' splice site and joins it

3. Lariat forms containing donor site and 2'-5' phosphodiester linkage

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Splicing with snRNAs

Back 65

1. U1 binds to 5' splice site

2. U2 binds to branch site in intron, bringing intron together with U1 and other snRNAs

3. U2 and U6 allow branch site A to attack 5' splice site

RNA molecule plays a key role in alignment/catalysis, and ATP unwinds, releasing RNPs

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Types of proteins that use splicing to be formed

Back 66

Tropomyosin

Calcitonin

Front 67

Marfan's Syndrome

Back 67

Splicing defect with fibrillin leads to defects in microfibrils and tissue around the heart

The person has chest pain, dyspnea, aorta weakness and blood seepage

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Marfan's Syndrome--why does the person have arachnodactyly too?

Back 68

Mutated fibrillin looks like a growth factor and binds to TGF-beta to cause growth. TGF-beta receptors need to be blocked with antibodies in Marfan's syndrome

Front 69

Systemic lupus erythematosus

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Splicing disease

Antibodies to U1 are formed, so splicing can't occur

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Globinopathies

Back 70

Almost all globin gene issues are due to a splicing problem

Front 71

Spinal muscle atrophy

Back 71

Mutation of exon 7 in the SMN-1 gene makes motor neurons more likely to die

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Becker muscular dystrophy

Back 72

Exon 27 in dystrophin gene; that dystrophin protein can't be made, decreases muscle fiber formation

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Frontotemporal Dementia with Parikinsonism

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Mutant exon 10 of Tau microtubule=abnormal filamentous structures in brain