Notes

Cell Cycle Checkpoints:
Checkpoints are needed to repair damaged chromosomes before they are duplicated. G1 checkpoints prevent entry into S phase, G2 prevents entry into Mitosis. Mitotic checkpoint prevents mitotic exit until all chromosomes are aligned.
p53:
- tumor suppressor gene/transcription factor that increases when cell is under stress. Enters nucleus where it binds to upstream sequences of genes. Can trigger cell response like apoptosis, cell cycle arrest, DNA repair
-p53 stimulates expression of MDM2, which inhibits p53 activity by blocking transcriptional activity, promotion of p53 nuclear export, and p53 degradation.
G1 DNA Damage Checkpoint
- DNA damage leads to phosphorylation and stabilization of p53 transcription factor --> increased activity --> increased transcription of downstream targets including p21
-p21 is a CKi that inhibits G1/S-Cdk and S-Cdk causing cell cycle arrest. Also stimulates Puma which results in apoptosis .
-393 amino acid residues with many domains. Tetramerization domain allows it to bind DNA.
-TP53 is most common mutated gene in cancer. Lots of mutations in DNA binding domain. 2 types of mutations: structural and ones that affect amino acid-DNA contact
G2 checkpoint blocks entry into Mitosis:
-Active Cdk1/Cyclin B controls entry into mitosis
-DNA damage signals active protein kianse ATM and Chk1 that phosphorylate and inactivate Cdc25, blocks entry into mitosis. Signal turned off when DNA is repaired.
Chromosomes attach to kinetochore MTs via centromere by cohesion. Cohesion is cleaved by separase. Attachment to centromere relies on build up of proteins.
APC triggers sister-chromatid separation
-Anaphase promoting complex is an E3 ubiquitin ligase. Requires Cdc20. Securin is substrate so chromosomes can be cleaved
- Tension needs to be sensed to satisfy checkpoint and trigger APC
Dysfunction leads to aneuploidy.

DNA Damage and Repair:
Types of DNA Damage: errors in DNA synthesis, spontaneous/endogenous source, exogenous sources (carcinogen, UV light)
Rate of DNA damage: 60,000 damaging events per day
150 genes encode DNA repair proteins.
Types of DNA mutations:
- point mutations: alteration in a single. base pair and small deletions that directly affect function of one gene. Missense is a change in a single amino acid within a protein. Nonsense is a single mutation that codes for a stop codon. Frameshift involve the addition or removal of one nucleotide shifting the codon reading frame
- chromosomal abnormalities involve alterations in large segments of DNA. Inversion: segments switch. Deletion: complete loss of region. Translocation: one part of a region moves to another chromosomes. Insertion: one part of a chromosome added to another.
Errors in DNA replication cause spontaneous mutations. Arise in regions of DNA containing tandemly repeated sequences (GTC). Portion of the newly synthesized strand loops out into a single stranded form, resulting in the same region being copied twice, adding 9 nucleotides.
Depurination and deamination:
-Depurination: removal of purine base (guanine)
-Deamination: removal of amino group from base. Converts cytosine to uracil. CG bp made into UA (transition mutation).
-Deamination of DNA nucleotides: Cytosine --> uracil, adenine --> hypoxanthine, guanine --> xanthine, 5-Methylcytosine --> thymine.
Bulky adducts: thymine residues in the same DNA strand become covalently attached. Distorts DNA.
DNA alkylation: transfer of alkyl group from one molecule to another. Methyl or ethyl groups are transferred to reactive sites on bases and to phosphates in DNA backbone. Environmental carcinogens are electrophiles that act by alkylation DNA.
Alkylation of N7 position of guanine: miscoding through abnormal base-pairing. Depurination of guanine residues leads to strand breakage. Cross linking of DNA occurs.
Reactive oxygen species: give rise to oxygen with an unpaired electron. Can arise from oxidative respiration. Guanine most vulnerable to ROS due to lowest reduction potential. Gives rise to point mutations.
DNA Repair pathways:
Base Excision Repair:
- cytosine deamination leads to C-G --> A-T transition
- Uracil DNA glycosylase removes U, leaving no base
- AP endonuclease cleaves sugar with no base, so full nucleotide is removed
- DNA Poly. and DNA ligase add nucleotide to seal nick.
Nucleotide Excision Repair:
- activated by pyrimidine dimers
- nuclease cleaves phosphodiester backbone of abnormal strand on both sides of sitrotion
- oligonucleotide containing lesion is pealed away from DNA by DNA helicase.
- large gap is produced in DNA helix that is repaired by DNA Poly. and DNA ligase.
Mismatch Repair
- DNA mismatches produced during replication, HR, cytidine deamination or other DNA replication, cytidine deamination
-MSH2-MSH6 heterodimers bind to single base-pair mismatches.
- MLH1, PMS2, EXO1 recruited to complex
- lesion dgiested by EXO1, and filled in by DNA polymerase
- proliferating cell antigen links the DNA Polymerase to DNA template.
Repair for DNA Double Strand Breaks:
- 5'-3' exonuclease activity of RAD50/MRE11/NBS1 exposes both 3' ends
- RPA coats single stranded DNA
- Assembly of BRCA1, BRC2, and RAD51
- RAD51 mediated homology search of intact sister chromatid DNA
- Strand invase, DNA synthesis and ligation
Xeroderma pigmentosum: inability to repair DNA damage from UV light, autosomal recessive
Hereditary non-polyposis colorectal cancer: inactivation of DNA mismatch repair proteins resulting in few adenomas and early colorectal cancer
BRCA1 and BRCA2 participate in double strand repair pathway. Women who inherit one mutant allele of BRCA1 or BRCA2 tumor suppressor gene have a high chance of developing breast cancer.

Circadian Clock:
Circadian clock is endogenous cellular mechanism for keeping track of time. Enable organism to anticipate daily environmental changes and tailor behavior and physiology to appropriate time of day. Clock is synchronised to day-night cycle. Disruption of circadian rhythms impacts many physiological events. Natural selection favors organisms with internal biological timer.
Circadian regulates genes involved in sleep pattern, feeding behavior, hormone release, BP, body temp., metabolism, cognitive performance
Circadian clock in mammals
- central oscillator in the brain (suprachiasmatic nucleus in hypothalamus) and peripheral oscillators in all cells of the body. Association between central oscillator and visual part of light because light acts as environmental cue.
Food behavior and exercise affect circadian clock. Organs more active during the day cause of eating and day time activity. Melatonin secretion increased during night. Disruption can cause disease.
Circadian clocks are derived from sunlight:
- light detected by photoreceptors of retina
- light info conveyed to SNc where it entrains an intracellular molecular clock consist of positive elements; BMAL1 and CLOCK and negative elements; PER and CRY.
MoA of circadian clock system:
- CLOCK and NPAS2 are transcription factors that form heterodimer with BMAL1
- bind to DNA sequence on E box enhancing transcription of clock genes; PER and CRY
- PER and CRY form a dimer as proteins
- Acts on BMAL1;CLOCK(NPAS2) to repress the transcription of PER and CRY.
- PER and CRY proteins are gradually degraded through post-translational modifications
- cycle repeats
PER and CRY phosphorylation and ubiquitination.
- kinases, casein kinase 1 phosphorylate the PER and CRY proteins
- promotes polyubiquitination by their respective E3 ubiquitin ligase complexes that tag proteins for degradation by 26S proteasome complex
- ensure a required temporal delay of negative arm of TTFL
Liver cells can enter cell cycle after hepatectomy, result is restoration of liver mass in a few days
- removal of CRY results in impaired regeneration
- Wee1 expression rapidly increased after hepatectomy. Wee1 promoter contains 3 E boxes which regulate mitotic entry. BMAL1 and CLOCK can bind to E boxes.

Stem cells and potential stem cell therapies.
- A stem cell is an unspecialized cell which can give rise to 250 specialized cells in the body. Under go asymmetric cell division and can renew itself.
Mature stem cells: adult cells obtained from mature body tissue like embilical cord and placenta, are multipotent giving rise to limited cell types
Early stem cells: embryonic and blastocystic cells obtained from inner cell mass of blastocyst, are pluripotent and give rise to all cell types in the body.
Zygote is totipotent. Blastocyst is pluripotent. Granulation layers are multipotent.
Classification of Haematopoietic stem cells and progenitor cells
- Long term HSC: rare cells, mainly rest with limited number of cycling cells
-Short term HSC: contribute to short lived cell populations for limited period of time, less than 12 weeks
-Progenitor cells: downstream of stem cells give rise to single lineage such as T and B cells, NK cells. Short life span.
Mesengenic process: proliferation --> commitment --> lineage progression --> differentiation --> maturation
Neural stem cells in the brain give rise to three major cell types: neurons, astrocytes, and oligodendocytes.
Differentiation pathways of adult stem cells:
- hematopoietic stem cells give rise to all types of blood cells
- bone marrow stromal cells give rise to bone cells, cartilage cells, fat cells
- neural stem cells give rise to three major cell types
- epithelial stem cell give rise to lining of digestive tract and several other types
- skin stem cells are in the basal alyer of the epidermis and give rise to keratinocytes. Follicular stem cells give rise to hair follicles and epidermis
Cancer stem cells can renew and give rise to more cancer cells
Stem cells could be used to fight diseases like leukemia but problems with immune rejection and HSCs are difficult to isolate.
Somatic Cell Nuclear Transfer: Mammary gland cells of 6 year old female sheep isolated and cultured and then inserted into oocyte, Dolly the sheep developed
Culture of embryonic stems cells isolated from blastocyst
Autologous tissue grafts may be able to be generated by fusing cell biopsy from a patient with enucleated oocyte to produce tissue.
Embryonic stem cells were able to be used to develop into insulin producing pancreatic cells for Type 1 diabetes patient
Gene expression during differentiation is the key to understanding how cells are differentiated. For example, Oct4 is expressed in inner cell mass and in culture embryonic cells but not in differentiated cells.