Cell lectures 10, 11, 12, 13

* base substitution
* purine to purine or pyrimidine to pyrimidine

* base substitution
* purine to pyrimidine or pyrimidine to purine

* type of spontaneous DNA damage
* shift of proton position in a nucleotide
* keto (A) --> enol (G)
* amino (T) --> imino (C)

* type of spontaneous DNA damage
* removal of an amine group and replaced with an oxygen molecule
* cytosine --> uracil
* adenine --> hypoxanthine
* gaunine --> xanthine
* 5-methylcytosine --> thymine

* type of spontaneous DNA damage
* removal of a purine base (A or G) from the deoxyribose sugar by the hydroloysis of the N-glycosidic bond
* what remains is called the apurinic site (lack of purine and -OH group left in its spot)

* type of spontaneous DNA damage
* oxidation of deoxyguanosine (dG) --> 8-oxo-deoxyguanosine (8-oxo-dG)
* oxidation and subsequent deamination of deoxy-5-methylcytosine --> deoxythymidine glycol

* x-rays and radioactive radiation
* results in chemical alterations to DNA and/or double stranded breaks

* can cause the formation of a covalent bond between to adjacent thymidines to form a dimer

* type of chemical that causes DNA damage
* erroneouslly incorporated into DNA
* ex. 5-bromouracil

* type of chemical that causes DNA damage
* attache alyl group
* guanine --> O6-methylguanine

* type of chemical that causes DNA damage
* causes deamination
* see deamination card
* ex. nitrous acid

* type of chemical that causes DNA damage
* agent that inserts itself into stacked DNA to cause a frameshift mutation
* ex. ethidium bromide, proflavin

* benzo(a)pyrene from cigarettes oxidizes in the cell and then covalently bonds to guanine in DNA thereby disrupting the C-G bonds and distorting the double helix structure
* its not a carcinogen until oxidized

* DNA photolyase breaks the bond within the thymine dimer
* the enzyme is only present in prokaryotes

* transfers the methyl group from the O6-methylgaunine (which had been formed from guanine by an alkylating agent) to a cystein residue on the methyltransferase active site

* types of repair mechanisms that always include
- recognition
- removal
- replacement
- rejoin
* ex base excision, nucleotide excision, and mismatch repair

* single damaged bases are repaired
* DNA glycosylase recognizes the damaged base and breaks the N-glycosidic bond resulting in an AP site
* an AP endonuclease cleaves the phosphodiester backbone adjacent to the AP site
* the remaining deoxyribose is removed by a phosphodiesterase
* DNA polymerase synthesizes the DNA
* DNA ligase seals the nick

* repairs bulky lesions
* distortion in the double helix is recognized by a protein complex containing XP-C and 23B. The transcription factor TFIIH is recruited
* TFIIH uses its helicase activity to unwind the DNA. XP-G and RPA bind to the complex and help to further unwind and destabilize the DNA until a bubble of ~25 bases forms
* XP-G and XP-F use their endonuclease activity to cut the DNA strand at the 5' and 3' ends respectively. Removes piece ~30 bases long
* DNA filled in by DNA polymerase and nick is sealed by ligase

* RNA polymerase (during transcription) is stalled by DNA damage and recruits CsA and CsB
* CSA/CsB recruits XPA, RPA, and TGIIH
* TGIIH unwinds DNA and XPG and XPF are recruited to cut and remove the piece of DNA
* gap filled by DNA polymerase and sealed by DNA ligase

* in E. coli, A residues in a GATC sequence are methylated by DAM methylase to form 6-methyladenine
* DNA polymerase only incorporates A (not CH3-A) so for a short time after replication only the parent strand has the methyl residues
* during this lag time there is the ability to recognize the correct strand
* MutL-MutS binds to the mismatched pairs. MutL connects MutS with a nearby MutH which can distinguish methylated parental strand from unmethylated daughter strand. DNA on both sides of the mismatch is threaded through MutL-MutS complex, creating a DNA loop.
* the endonuclease activity of MutH becomes activated when the complex encounters a hemimethylated GATC sequence
* MutL and MutS act together with DNA helicase, SSB, and single-strand excise the DNA between the strand break and the mismatch
* DNA polymerase and ligase fill and seal

* A complex of MSH2 and MSH6 proteins binds to mispaired DNA in such a way to distinguish the template and newly synthesized daughter strands.
* this triggers binding of MLH1 endonuclease which cuts the daughter strand
* a DNA helicase unwinds the helix and an exonuclease removes several nucleotides, including the misincorporated base
* DNA polymerase and ligase fill and seal

* repair of double strand breaks utilizing the sister chromatid
* error free

* Double strand break sealed without sister chromatid to fill in the blank
* loss of nucleotides due to degradation at ends
* error prone

* A rare autosomal recessive genetic disorder.
* Patients show dry skin (xeroderma) and many freckles (pigmentosum) in sun-exposed area of skin.
* Extremely sensitive to UV radiation.
* Affected individuals have increased risk of skin cancers early in life.
* Deficient in nucleotide-excision repair.
* Inherited defects in any one of the XP genes (XPA through XPG

* Rare recessively inherited disorder
* Presents with growth retardation and neurological degeneration
* Defect in transcription-coupled repair (CSA, CSB)

* A dominantly inherited syndrome also known as Lynch syndrome (Henry Lynch).
* Increases the chance of colon cancer.
* Affected individuals have 80% chance of developing colon cancer by the age of 65.
* The number of colon polyps is much less compared to familial adenomatous polyposis (FAP), another form of rare inherited form of colon cancer.
* Increases the risk of other cancers, including endometrium and ovary.
* Defect in mismatch repair. Mutation in MLH1 or MSH2 is most common.

mitosis and cytokinesis yield two daughter cells

terminally differentiated cells withdraw from cell cycle indefinitely
* a cell returning from G0 enters at early G1 phase

* RNA and protein synthesis.
* No DNA synthesis

* within G1
* a cell that passes this point is committed to pass into S phase

* DNA synthesis doubles the amount of DNA in the cell
* RNA and protein also synthesized

* no DNA synthesis
* RNA and protein synthesis continue

* Induced by growth factors or mitogens early in G1 phase
* Determine whether a cell should divide in response to external stimuli
* cyclin D (three types) + Cdk4 or Cdk6
* D cyclins induced by growth factors

* Expressed late in G1
* Required for the passage through the restriction point.
* cyclin E + Cdk2

* Induced at late G1 after cyclin E
* Required for initiation of DNA replication
* cyclin A + Cdk2, Cdk1

* Appears at the beginning of G2 and accumulates through G2
* Required for the entry into mitosis
* cyclin B + Cdk1

Thr-14 and Tyr-15: Inhibitory phosphorylation:
* Phosphorylated by Wee1 (Y15) and Myt1 kinases (Y15 and T14)
* Dephosphorylated by Cdc25A and Cdc25C

Thr-160 or 161: Activatory phosphorylation
* Phosphorylated by Cdk-activating kiinase or CAK

* Inhibits Cdk4
* p15, p16, p18 and p19
* Inhibits G1-CDK

* p21, p27 and p57
* Inhibit G1/S-CDK & S-CDK

-Hypophosphorylated Rb associates with E2F and represses its transcriptional activity.
-in response to growth factors, D type cyclins are synthesized and activate G1/CDK which phosphorylates Rb.
-Phosphorylated Rb cannot bind to E2F.
-E2F transcribes genes necessary for S phase transition.
-Activation of E2F leads to increased expression of G1/S cyclin (cyclin E) and S cyclin (cyclin A) causing activation of G1/S CDK or CyclinE-CDK2.
-G1/S-CDK can further phosphorylate Rb even when growth factors are removed and D-type cyclin levels fall.

-In early G1 phase, G1/S CDK or cyclin E/CDK2 complex is inhibited by the CDK inhibitor p27.

-After cells pass through restriction point, E2F-dependent transcription of cyclin E results in activation of cyclin E/CDK2.
-Cyclin E/CDK2 phosphoryates p27 and leads to its degradation.
-Activation of cyclin E/CDK2 leads to activation of MCM helicase and initiation of DNA replication.

* chromosome duplication and cohesion
* centrosome duplication

Chromosome condensation
Centrosome migration
Mitotic spindle assembles

Nuclear envelope breakdown
Chromosomes attach to spindle microtubules via kinetochores

Chromosomes move to spindle equator and are aligned at the metaphase plate

Centromeres split
Sister chromatids separate

Daughter chromosomes arrive at the spindle poles
Nuclear membranes reform
The spindle disappears
Chromosomes decondense

* reformation of interphase microtubule array
* contractile ring forms cleavage furrow

* chromatin condensation: phosphorylation of condensins
* nuclear envelope breakdown: phosphorylation of lamins, nuclear pore complexes, and inner nuclear membrane proteins
* fragmentation of golgi apparatus: phosphorylation of golgi matrix proteins
* spindle formation: phophorylation of centrosome and microtubule-associated proteins

* Prevents initiation of mitosis until DNA replication is complete
* ATR associates with replication forks and activates Chk1 kinase
* Chk1 phosphorylates and inactivates Cdc25C, thereby inhibiting entry into mitosis.

* Tumor suppressor gene product
* Highly unstable and is present at very low concentration under normal condition
* Activated in response to DNA damage
* Transcription factor
* Induces p21, a CKI which inhibits G1/S-CDK and S-CDK
* also promotes apoptosis via the mitochondrial pathway (tumor suppressor)

* Blocks progression into S phase.
* ATM and ATR kinases are activated in response to DNA damage and phosphorylate Chk2 and Chk1 kinases.
* ATM, ATR, Chk1 and Chk2 can phosphorylate and stabilize p53.
* p53 induces transcription of p21 which inhibits G1/S-Cdk and S-Cdk.
* Rb hyperphosphorylation is prevented causing cell cycle arrest.

* Damaged DNA activates ATM and ATR protein kinases which activate Chk2/Chk1 kinases.
* Chk1/Chk2 phosphorylate Cdc25C leading to its cytoplasmic sequestration and subsequent inhibition of M-Cdk (bc its sequestered and not there to activate M-Cdk?)
* ATM/ATR also phosphorylate and stabilize p53 causing induction of p21 and inhibition of Cdks.
* M phase entry is prevented.

* translocation of this from the inner leaflet of the plasma membrane to the outer membrane signals a phagocytic cell to consume it
* apoptosis signal

* caspases get activated during apoptosis and cleave ICAD off of CAD thereby activating it to degrade DNA

* 8 and 9
* contain long pro domain
- death effector domain (DED) - caspase - 8
- caspase recruitment domain (CARD) - 9

* 3, 6, 7
* short pro domain

* a family of cyseine proteases that cleave after Asp residues
* Synthesized as inactive proenzymes that contain 3 domains: NH2 terminal pro-domain, large subunit and small subunit.

* Anti-apoptotic Bcl-2 family proteins:
- Bcl-2, Bcl-xl, Mcl-1
- The Bcl-2 family proteins regulate mitochondrial integrity and
cytochrome c release
* Pro-apoptotic Bcl-2 family proteins:
- Bax, Bak, Bid, Bim

* mitochondrial
* The major mechanism of apoptosis triggered by various physiological and pathological stimuli
* Mitochondrial integrity is compromised causing release of proapoptotic molecules
* Cytochrome c released into the cytoplasm initiates apoptosis
* The Bcl-2 family proteins regulate mitochondrial integrity and cytochrome c release
* Cytochrome c facilitates interaction of apoptosis promoting activating factor (Apaf 1) with initiator procaspase-9 through CARD domain, causing activation of caspase-9.
* Caspase-9 activates its downstream effector caspases, such as caspase-3/-7 and triggers caspase cascade.

* Binding of death ligand to the receptor triggers receptor oligomerization
* DD of the receptors recruits other DD-containing adapters (e.g., FADD, TRADD)
* FADD not only contains DD domain but also DED domains.
* FADD can interact with initiator caspase-8 or FLICE (FADD-like ICE) through DED to form death-inducing signaling complex (DISC)
* Binding of initiator caspase (e.g., caspase-8) to DISC causes autoproteolytic activation
* Initiator caspase can then cause activation of its downstream effector caspase, such as caspase-3 and trigger caspase cascade

* apart of the Bcl-2 family
* allows for cross talk between intrinsic and extrinsic cell death pathway
* Caspase-8 can cleave the Bcl-2 family protein Bid
* The truncated Bid can translocate to the mitochondria and facilitate release of cytochrome c
* Thus, cleavage of Bid can amplify cell death by the extrinsic pathway via the mitochondrial pathway

* Catalytically inactive version of caspase-8
* High level of c-FLIP can inhibit extrinsic cell death pathway by inhibiting apoptosis

* Inhibit mitochondrial cell death pathway by inhibiting caspase-9 and -3.