Molecular, carcinogenesis, Familial disorders, viruses, DNA repair, telomeres, Cell cycle Flashcards
What is the definition of RNA interference?
RNAi = The process of mRNA degradation that is induced by dsRNA in a sequence specific manner
Can specifically silence the expression of any gene for which the sequence is available
Large set of specific cellular responses to RNA called RNA silencing
Group of proteins degrade ds mRNA, meant to protect genome from viruses that use dsRNA in their life cycles
What is the process of RNAi?
First, Drosha and DGCR8 complex process miRNA in the nucleus to form pre-miRNA. This is exported to the cytoplasm by Exportin 5. Dicer then processes it into mature ds-miRNA (siRNA), which is 18-25 nucleotides in length. RNA strands are separated
miRNA is incorporated into an RNA-induced silencing complex (RISC). miRNA guides RISC to the 3’ untranslated region of the complementary mRNA and represses expression. (such association can result in either the inhibition of translation or the mRNA or its degradation, or both)
What are miRNAs? Where are the transcribed from?
RNA silencing plays a critical role in regulation of cell growth and differentiation using endogenous small microRNAs (miRNAs)— mostly transcribed from introns or other noncoding areas of the genome; +/- exons of other genes
What is siRNA?
Long dsRNA molecules are digested by Dicer into 19-21(18-25) nucleotide fragment (siRNA= mature miRNA)
T/F? Loss of Dicer enzyme has been associated with cancer progression.
True
How do you interpret Western blots?
No bands present= Negative
Bands present= Positive
Bands present, but pattern does not meet criteria for positivity= Indeterminate
What is FISH?
What does it require to run?
What chromosomal abnormalities can it commonly detect? List examples of this.
What phase of the cell cycle is it performed on?
form of cytogenetic analysis (detects chromosomal abnormalities)
o Requires knowledge of DNA sequence to create a specific probe
o Can assess chromosomes for amplifications, deletions, or other abnormalities
o Uses: determine HER2 status, detection of N-myc amplification, etc.
o Can be performed on interphase nuclei
What is comparative genomic hybridization (CGH)?
What chromosomal abnormalities can it commonly detect?
What chromosomal abnormalities can it NOT detect?
How does it differ from FISH?
CGH was more recently replaced by what test, which can better detect abnormalities of a smaller magnitude?
Also a form of cytogentic analysis (detects chromosomal abnormalities)
o Detects increases or decreases in cancer DNA relative to controls (changes in copy #)
Cannot determine the cause (amplification vs. translocation, etc.)
Balanced rearrangements (inversions, translocations) may escape detection
o If sequence is unknown, cannot select a probe for FISH – CGH is a more generalized technique
o More recently replaced by microarrays, which can detect abnormalities of a smaller magnitude
What is the difference in what annexin dye vs propidium iodide stains for or detects in regards to apoptosis in cells?
o Annexin dye = stains cells that have initiated early apoptosis; detects PS bound by annexin V protein
Binds phosphatidyl serine on the outer leafleft of cells undergoing early cell death
o Propidium iodide = detects cells in late apoptosis because it requires permeability to the nucleus
Binds DNA of cells if is able to enter owning to damaged membranes, hence indicating late cell death
Dot plot with annexin on x axis and PI on y axis – determine which answer choice is correct with percentage of cells in necrosis/late apoptosis, early apoptosis, or overall apoptotic
Sorry no picture- use your imagination
* see example pictures in Phoenix
Y axis= PI Necrosis
Viable cells Apoptosis
X axis= Annexin
In regards to the process of carcinogenesis, describe from the list below which does NOT and which DOES lead to formation of a tumor?
- A single low dose of initiator
- Promoter is applied only or promoter is applied before initiator
- Low doses of initiator followed by repeated doses of promoter
- Application of promoter is delayed for several months after initiator
- A single low dose of initiator does not give rise to a tumor
- No tumor develops if only promoter is applied or if promoter is applied before initiator
- Low doses of initiator followed by repeated doses of promoter gives rise to a tumor
- Tumor can develop if application of promoter is delayed for several months after initiator
As part of the steps of carcinogenesis, what is tumor initiation?
Tumor initiation: involving the interaction of a reactive chemical species with DNA to produce damage, which if not repaired before the next cell division, would lead to erroneous DNA replication resulting in fixation of mutations within the genome of individual cells
Initiator: generates mutation but, by itself, does not cause tumor formation
As part of the steps of carcinogenesis, what is tumor promotion?
Tumor promotion: clonal expansion of an initiated cell as a consequence of events that alter gene expression, so as to provide the cell with a selective proliferative advantage
o Promoter: exerts nongenetic effects, but triggers proliferation
Increased division increases likelihood that a second mutation will occur
As part of the steps of carcinogenesis, what is tumor progression?
Tumor progression: the stage whereby benign lesions acquire the ability to further grow, to invade adjacent tissues, and to establish distant metastasis
o Progression: development of a 2nd mutation
Increased genomic instability and karyotypic alterations
Acquires ability to grow, invade, metastasize
Which of the steps of carcinogenesis is reversible
tumor promotion
Which proteins or mutations that occur as cancer associated genetic changes are responsible for regulation of cell proliferation, death, and differentiation?
T/F: Mutations of these proteins correlate with the highest risk of cancer?
Gatekeeper mutations
o Typically tumor suppressor genes
o Mutations of these proteins correlate with the highest risk of cancer= TRUE
Which proteins or mutations that occur as cancer associated genetic changes are responsible for guarding the integrity of the genome?
Loss of function increases risk of what?
These mutations often involve what part of cell functions?
Care taker mutations
Loss of function increases the risk of acquiring gatekeeper mutations that would lead to oncogenic growth
o Often involve DNA checkpoints and repair factors (i.e., BRCA1/2 mutation)
Which proteins or mutations that occur as cancer associated genetic changes alter the tumor microenvironment?
landscaper mutations
List the original 6 Hallmarks of cancer.
- Sustained proliferative signaling
- Evading growth suppressors
- Activating invasion and metastasis
- Enabling replicative immortality
- Inducing angiogenesis
- Resisting cell death
List the enabling Hallmarks of cancer.
- Genomic instability and mutation
2. Tumor promoting inflammation
List the emerging Hallmarks of cancer.
- Reprogramming of E metabolism
2. Evading immune destruction
The following are examples of which of the hallmarks of cancer?
Upregulation of EGFR or HER2/neu
Activating mutations in BRAF
Activating mutations in catalytic subunit of PI3K
Upregulation of CDK4
Loss of negative feedback via:
• Activating mutations in RAS
• Loss of function mutations in PTEN
• Activation of mTOR
Sustained proliferative signaling
The following are examples of which of the hallmarks of cancer?
Loss of E-cadherin
Upregulation of N-cadherin
Mutation/inactivation of B catenin genes
Increased ezrin
Upregulation of TFs involved in EMT: Snail, Slug, Twist, Zeb 1/2
Upregulation of protease genes
Activating invasion and metastasis
The following are examples of which of the hallmarks of cancer?
RAS and c-Myc can upregulate expression of angiogenic factors
Loss of VHL (results in upregulation of VEGF)
Upregulate VEGF and/or FGF expression
Loss of TSP-1 or IFN-B
Inducing angiogenesis
The following are examples of which of the hallmarks of cancer?
Defects in caretaker genes (DNA repair machinery)
Loss of telomeric DNA
BUB1
Genomic instability and mutation
The following are examples of which of the hallmarks of cancer?
Warburg effect (aerobic glycolysis)
Upregulation of GLUT1 to include Glc delivery to cytoplasm
Increased HIF-1a- upregulates glycolysis
Gain of mutation mutations in isocitrate dehydrogenase (IDH1/2)
Reprogramming of E metabolism
The following are examples of which of the hallmarks of cancer?
Loss of RB (gatekeeper)
• Down-regulation of TGF-B receptors (TGF-B prevents phosphorylation of RB)
• Loss of SMAD4 (transmits signals from TGF-B)
• Deletion in p15INK4B
• CDK4 may be unresponsive to inhibitory signals from p15INK4B
o Therefore, Cyclin D-CDK4 complex can inactivate (hyperPhos) pRb
• Mutation in pRb itself
• Sequestration of pRb by viral oncoproteins (e.g., HPV E7)
Loss of P53 (another gatekeeper)
• Li-Fraumeni syndrome
• HPV E6 oncoprotein
Avoid differentiation
• Overexpression of c-Myc
Loss of contact inhibition
• Loss of Merlin, the cycoplasmic NF2 gene product
• Suppression of LKB1, which maintains epithelial polarity (normally overrules effects of Myc)
Redirection of TGF-B pathway (antiproliferative activates EMT)
Evading growth suppressors
The following are examples of which of the hallmarks of cancer?
TERT expression or upregulation of ALT – resistance to senescence and crisis/apoptosis
Loss of P53 – survive telomere erosion and BFB cycles
Enabling replicative immortality
The following are examples of which of the hallmarks of cancer?
• Upregulate factors that inhibit apoptosis = Bcl-2, BCL-XL, BCL-W, MCL-1, A1
o Activation of PI3K-AKT/Pkb pathway
• Downregulate factors that trigger apoptosis = Bax, Bak, Bim, Bid
o Loss of P53 (normally upregulates Bax expression)
o Loss of caspases
o Loss of Fas
Upregulation of survival factors (i.e., IGF-2, IL-3)
Evasion of autophagy
• PI3K, AKT, and mTOR inhibit autophagy
• Loss of Beclin-1, a BH3-only protein (normally triggers autophagy)
Necrosis may trigger inflammation to further tumor progression
Resisting cell death
The following are examples of which of the hallmarks of cancer?
Bioactive molecules supplied to the microenvironment, including GFs that sustain proliferative signaling, survival factors that limit cell death, proangiogenic factors, ECM-modifying enzymes to facilitate angiogenesis, invasion and metastasis, and induction signals that lead to activation of EMT
Release of ROS that are actively mutagenic
IL-1B production
Tumor-promoting inflammation
The following are examples of which of the hallmarks of cancer?
Deficiencies in development or function of CD8+ or CD4+ T cells, or NK cells
Secretion of TGF-B, which is immunosuppressive
Recruitment of Tregs, MDSCs, TAMs
ICAM-1 production
Evading immune destruction
Which of the following mechanisms of action is/are associated with loss of heterozygosity?
- Gene conversion
- Mitotic recombination
- State of possessing two identical forms of a particular gene
- Loss of chromosomal segment resulting in hemizygosity
- Degradation of a set of short-lived chromosomes, enabling cellular regulation at the level of chromosome instability
- Nondisjunction- loss of chromosome
o (1) Gene conversion: when the DNA strand is elongated, it temporarily switches templates and forms a hybrid with the cDNA strand of the homologous chromosome. It then reverts back to its original template (copy choice). If the cDNA strand contains an inactive allele, then LOH has occurred.
More frequent that mitotic recombination.
o (2) Mitotic recombination (recombination between sister chromatids)
o (3) Loss of chromosomal segment, resulting in hemizygosity (only single copy)
o (4) Nondisjunction: inappropriate chromosomal segmentation during mitosis may result in loss of an entire chromosome: If resulting daughter cell is triploid, then it will shed the extra chromosome (may result in LOH).
At the chromosomal level, what are the exact mechanisms of loss of heterozygosity?
Loss of heterozygosity (LOH) is defined as the loss of one parent’s contribution to the cell, can be caused by direct deletion, deletion due to unbalanced rearrangements, gene conversion, mitotic recombination, or loss of a chromosome (monsomy).
Which of the following is the most carcinogenic? aflatoxin B1
benzo[a]pyrine
4-aminobiphenyl
2-naphthylamine
o Alfatoxin B1 is produced by Aspergillus sp. and causes HCC with concurrent p53 mutations
o Characteristic G-to-T transversion
What are common mechanisms that occur with hypermethylation of CpG areas within the cell?
• DNA methylation specifies which genes are actively transcribed or silenced
•CpG dinucleotides are abundant within the 5’ ends of some genes, typically including the promoter and first exon (CpG islands)
o DNA methyltransferase (DNMT) attaches methyl group to 5-carbon of cytosines within this sequence results in gene silencing
Choose which of the following statements is true regarding methylation of CpG islands.
- Cancer cells exhibit global hypermethylation, which results in genomic instability.
- Local hypermethylation of gene promoters silences tumor suppressor promoters.
- Oncogenes can be activated through hypomethylation
These statements are true:
- Local hypermethylation of gene promoters silences tumor suppressor promoters.
- Oncogenes can be activated through hypomethylation
This was false and should read as follows:
1. Cancer cells exhibit global hypomethylation, which results in genomic instability.
What are the physical effects of methylation of the CpG islands?
- Physically blocks binding of transcriptional activators to the promoter
- Binds methyl-CpG binding domain proteins (MBDs), which recruit HDACs to form heterochromatin (inactivate, tightly coiled)
T/F: Normally, methylation occurs in multiple sites throughout the genome to block activation/movement of transposable elements to prevent chromosomal instability.
True
Which is the type of covalent adduct that is induced by UV light?
> 60% of pyrimidine dimers are TT
__________________________________________
•UV light damages DNA by forming pyrimidine dimers (covalent bonds between two pyrimidines: TT, CC, or TC)
• 30% are CC
• Very stable, mutagenic compounds
o TT is more able to be repaired by DNA enzymes and therefore only weakly mutagenic
o CC dimers are harder to repair and often result in CC –> TT substitutions during replication
• XP = lack of NER enzymes, sensitivity to UV radiation because of pyrimidine dimers
Which molecular diagnostic tool are used to study loss of heterozygosity or copy neutral LOH (uniparental disomy or gene conversion) whereby one allele or whole chromosome is missing and the other allele is duplicated with pathological consequences?
SNP arrays
The following describes which SNP inheritance pattern in a pedigree chart?
Males affected almost exclusively
Single recessive gene on X chromosome will cause dz
Gene alteration transmitted from mother to son
Affected males cannot transmit to sons
X linked recessive
*see Phoenix for picture examples
The following describes which SNP inheritance pattern in a pedigree chart?
Pattern of transmission of the mutant phenotype from an affected male parent to ALL female offspring, and from an affected heterozygous female to 50% of ALL offspring
X linked dominant
*see Phoenix for picture examples
The following describes which SNP inheritance pattern in a pedigree chart?
an affected individual has one copy of a mutant gene and one normal gene on a pair of autosomal chromosomes
Autosomal dominance
*see Phoenix for picture examples
The following describes which SNP inheritance pattern in a pedigree chart?
a genetic condition that appears only in individuals who have received two copies of an autosomal gene, one copy from each parent
Autosomal recessive
*see Phoenix for picture examples
You are given that a germline mutation is causing a truncated TSG in a sarcoma.
You then have PCR products of a control, a bx from normal SQ area, and bx from sarcoma of the same patient.
Which PCR is consistent with PCR products from these three?
- Germ line mutation is present in chromosome and passed to offspring
- Somatic mutation is not incorporated into chromosome or passed to offspring, but is transmitted as cell divides
- PCR from normal SQ should be the same as the PCR from the tumor since it’s a germline mutation.
Which familial or human genetic mutation has an autosomal recessive mutation in ATM kinase?
Ataxia telangiectasia
What is the normal function of ATM kinase, which is why having a mutation would lead to problems?
ATM kinase normally
responds to dsDNA breaks
activates CHK2
phosphorylates p53 to avoid its destruction
phosphorylates Mdm2, preventing it from binding to p53
T/F: People with ataxia telangiectasia have a hypersensitivity to UV radiation?
FALSE: Hypersensitivity to ionizing but not UV radiation
Which familial or human genetic mutation results from germ-line mutations in MMR genes, which undergoes LOH to silence TSG?
MSH2 and MLH1 most commonly affected
MSH6 and PMS2 less frequently involved
Hereditary nonpolyposis colon cancer
Which familial or human genetic mutation results from inherited defect in any of 8 different genes involved in NER?
Xeroderma pigmentosum
People with xeroderma pigmentosum are sensitive to which form of radiation and why is this?
o Sensitivity to UV radiation, resulting in cutaneous SCC and basal cell carcinomas
Humans totally rely on NER pathway to remove UV-induced pyrimidine dimers
Which familial or human genetic mutation results from:
o Autosomal dominant p53 mutation, so humans are heterozygous
o Tumors from these patients have a LOH and absence of wild-type P53
Li-Fraumeni syndrome
o Characterized by early onset of mesenchymal and epithelial malignancies
What is the Von Hippel-Lindau protein?
o VHL is a TSG – involved in degradation of HIF-1a
Normal O2: proline hydrolases use O2 to oxidize proline residues on HIF-1a (requires presence of alpha-ketoglutarate) –> HIF-1a binds to VHL–> ubiquitination
Hypoxia: proline hydrolases can’t oxidize HIF-1a –>
escapes and increases transcription of VEGF, PDGF, TGF-a, EPO
o Deletion of VHL vs. mutation in HIF-1a binding pocket
Alteration of the VHL gene by mutation, loss of heterozygosity, and promoter methylation has been found to be important to which cancer pathogenesis?
renal cell carcinoma
Loss of VHL protein in renal cell carcinoma leads to constitutive activation of what?
HIF
Match the genes transcribed by HIF:
- Metabolism: choose the correct letter below
- Angiogenesis: choose the correct letter below
- Metastasis: choose the correct letter below
a. VEGF, PDGF, FLT-1
b. GLUT-1, GAPDH, PDK1, CA9, MT-4, LDH-A
c. LOX, VEGF-C, Met, CXCR4, CXCL12
o Metabolism: GLUT-1, GAPDH, PDK1, CA9, MT-4, LDH-A
o Angiogenesis: VEGF, PDGF, FLT-1
o Metastasis: LOX, VEGF-C, Met, CXCR4, CXCL12
What type of chromosomal translocation occurs in CML? (humans vs. dogs)
• Reciprocal translocation resulting from deletion in chromosome 22 with insertion in chromosome 9 (Humans)
Philadelphia chromosome or BCR-ABL–> puts ABL under control of BCR, resulting in constitutive activation
• Dogs: Raleigh chromosome – 9 and 26
In regards to comparative aspects, match the association of human viruses to the appropriate cancer. Viruses: Human T cell leukemia virus Human papilloma virus Epstein-Barr virus Human hepatitis B and C viruses Human herpes virus 8 Polyomaviruses
Cancers: Burkitt’s lymphoma Kaposi’s sarcoma Hepatocellular carcinoma T cell leukemia Cervical cancer Merkel cell carcinoma Nasopharyngeal carcinoma Primary effusion LSA (B-cell)
Human T cell leukemia virus: T cell leukemia
Human papilloma virus: cervical cancer
Epstein-Barr virus: Burkitt’s lymphoma, Nasopharyngeal carcinoma
Human hepatitis B and C viruses: hepatocellular carcinoma
Human herpes virus 8: Kaposi’s sarcoma, primary effusion LSA (B-cell)
Polyomaviruses: Merkel cell carcinoma
Which of the following is an RNA virus?
Human papilloma virus Epstein-Barr virus Human T cell leukemia virus Human hepatitis B and C viruses Human herpes virus 8 Polyomaviruses
human T-cell leukemia virus
- Human T-cell leukemia virus is an RNA virus (retrovirus) – 1-4% risk of adult T-cell leukemia
- Can be transmitted to offspring via milk
- Infects T-cells (typically CD4+) –> gains entry via GLUT1 transporter –> insertional mutagenesis
- Contains its own oncogene (Tax) – activates transcription of proviral DNA sequences and 2 growth factors (GM-CSF, IL-2)
T/F: In regards to the human papilloma virus, in benign lesions, viral DNA exists as an episome (non-integrated, circular form). In malignancies, it randomly integrates into chromosomes.
True
When the human papilloma virus causes malignancy, what is the main MOA that occurs as it randomly integrates into chromosomes?
This disrupts E2, which normally inhibits E6/7
Match the following oncoproteins encoded by the HPV virus with the mechanism of action: Virus: 1. E4 2. E5 3. E6 4. E7
MOA:
a. transforming protein that binds Rb
b. membrane protein that interacts with EGFR, PDGFR –
transforming protein that targets p53 for degradation
c. induces TERT expression
d. disrupts cytokeratins; important for viral release
e. overrides inhibitory effects of CDK inhibitors, p21 and p27–> entry to S
• E4: disrupts cytokeratins; important for viral release
• E5: membrane protein that interacts with EGFR, PDGFR
• E6: transforming protein that targets p53 for degradation
o Also induces TERT expression
o The disruption of p53 can result in increased levels of
p16, which is detectable with IHC
• E7: transforming protein that binds Rb (similar in structure to E1A protein of adenovirus)
o Also overrides inhibitory effects of CDK inhibitors, p21
and p27 –> entry to S
Which oncoprotein encoded by the papilloma virus causes SCC in cattle?
E5
Hepadnavirus (hepatitis B) causes hepatocellular carcinoma in which of the following species?
a. Bottle-nosed dolphin
b. Bearded dragon
c. Asian elephant
d. Woodchuck
e. Lavender ball python
Woodchuck
HBV causes cancer via (a) continuous cell proliferation to replace hepatocytes kill by HBV infectious cycles and (b) chronic inflammation, which serves as a tumor promoter
hepatitis B virus and HCC
Fill in the blank with ssRNA or dsDNA :
Hep B is a _____ virus (hepadnavirus) and Hep C is a _____ virus (flavivirus)!
Hep B is a dsDNA virus (hepadnavirus) and Hep C is a ssRNA virus (flavivirus)
Which tumor promotor (toxin) has synergistic effects with hepatitis B virus?
Alfatoxin B1 from Aspergillus is another tumor promoter; has synergistic effects with HBV
Answer T/F for each of the following questions regarding hepatitis B virus:
- L antigen binds to receptors on hepatocytes
- Integration of DNA into host chromosome is needed for replication
- Viral gene products may randomly insert into host chromosomal DNA
- Impairs NER (inactivates DDB1), directs E3 ligases to a variety of proteins
- True
- False- NO it is NOT needed
- True
- True
What is the most common oxidized base during DNA damage or the most common site of carcinogenesis?
8-oxo-deoxyguanosine
o Used as marker for overall oxidative DNA damage
o Problematic because new base can mispair with deoxyadenosine
o Results in original base pair (G:C) read as G:A results in T:A replicated strand
Example of a transversion
The degradation of p53 in normal cells is regulated by which protein?
MDM2
Which kinases are capable of phosphorylating p53 and preventing it from degradation by blocking MDM2 from binding?
• Protection from degradation: phosphorylation of p53 blocks Mdm2 binding
o ATM kinase, Chk1, and Chk2 can all phosphorylate p53
What process sets the phosphorylation of p53 into motion?
dsDNA breaks activate ATM kinase –> activates Chk2 kinase –> phosphorylates p53 to prevent its degradation
T/F: ATM kinase can also phosphorylate Mdm2, which inactivates/destabilizes it?
True
Mdm2 prevents p53-driven transcription by what 3 ways?
ARF binds Mdm2 to inhibit its action increases p53 levels
- Preventing p53 binding to p300/CBP, which activates transcription by histone acetylation
- Actively recruiting other enzymes that block p53-mediated transcription by methylating histones
- Ubquitination of p53, which exports it from the nucleus
Which 2 pathways activate MDM2?
Activated by PI3K pathway and Ras
PI3K –> AKT/PKB kinase phosphorylates Mdm2 at a different site than ATM kinase –> Mdm2 translocates to the nucleus to attack p53
Ras –> Raf –> MAPK–> Ets/AP-1 transcription factors increase transcription of Mdm2 gene
Which gene or protein inhibits MDM2?
Inhibited by p16ARF gene, which encodes ARF protein
The following questions address DNA repair mechanims:
- What are 2 main strategies in which cells have for detecting and removing normal, but miscopied nucleotides during DNA replication?
- What are 2 main DNA repair pathways that remove chemically altered bases?
- proofreading by DNA pol and MMR
2. BER and NER
Which DNA repair pathway is this?
corrects microsatellites (stuttering of DNA pol) and bulges/loops in double helix due to inappropriate bases
Pathway: MutS/MutL complex reached PCNA assoc. with replication fork. Loads EXO1, which resects the fork backward past the lesion. DNA pol δ and ε fill gap.
Mismatch repair
Microsatellites can be detected using gel electrophoresis; presence indicates MMR deficiency
Which DNA repair pathway is this?
DNA pol looks backward; if a copy error is detected then it uses its 3’-to-5’ exonuclease activity to move backward
DNA pol δ checks lagging strand, pol ε checks leading
pol κ (repairs 8-oxo-dg) and pol β (corrects lesions during BER)
Proofreading
Error prone polymerases that LACK proofreading
A deficiency in what DNA repair pathway causes hereditary non-polyposis colorectal cancer?
MMR
DNA base damage, occurring as a result of endogenous oxidative processes or exogenous DNA damage (ionizing radiation) is repaired by what pathway?
BER
DNA glycosylase recognizes abnormal base, cleaves its covalent bond to deoxyribose. Then:
Short patch: removes single nucleotide
• Occurs if glycosylase has β-capability
• APE1 removes flap. DNA pol β fills gap, PARP is
recruited to recruit ligase and seal the nick.
Long patch: removes 4-7 nucleotides
• Occurs if glycosylase lacks β-capability
• APE1 removes flap. PCNA, DNA pol δ and ε,
replication factor C create flap of 2-10
nucleotides. FEN1 nuclease cleaves overhang.
Nick sealed by ligase I.
Which DNA repair mechanism removes the following:
1. pyrimidine bases that produce 6-4 photoproducts
(6-4PPs)
2. cross links in the form of cyclobutane pyrmidine dimers
3. other bulky chemical adducts caused by exogenous agents
NER
A deficiency in the NER DNA repair pathway leads to which disease?
XP
Which DNA repair mechanism edits the template strand during active transcription?
Transcription-coupled repair
Removes 6-4-photoproducts (6-4PP) better than global genome repair
Which DNA repair mechanism addresses nontemplate strand of transcribed genes and nontranscribed regions of the genome?
Global genome repair
Removes cyclobutane pyrimidine dimers (CPDs) better than transcription-coupled repair
T/F: P53 affects global genome repair but not transcription-coupled repair
True
Which DNA repair pathway works in this 4 step process?
- recognition of damaged DNA
- excision of an oligonucleotide to 24-32 residues containing the damaged DNA by dual incision of the damaged strand on either side
- filling in of the resulting gap by DNA polymerase
- ligation of the nick
NER
- Mutations in non-homologous end joining lead to what disease process?
- Absence of non-homologous end joining leads to what disease process?
- NBS1 = Nijmegen breakage syndrome, a recessive disorder with chromosomal instability, radiosensitivity, and increased incidence of lymphoid tumors
- SCID
Where in the cell cycle does NHEJ take place?
operates throughout the cell cycle (esp. G1)
error-prone
Where in the cell cycle does HR take place?
Occurs during S and G2
when there is a newly replicated sister chromatid available as a template – high fidelity repair due to availability of a template
What TSG has been found to display LOH and observed in the HR DNA repair pathway?
BRCA1/BRCA2
- How do BRCA2 and RAD51 interact during HR?
- BRCA2 mutation occurs in which exon?
- BRCA2 mutation prevents what in regards to its interaction with RAD 51, leading to failure in which DNA repair pathway?
- BRCA2 contains multiple direct binding sites for RAD51
- Exon 11 BRCA2 mutation
- RAD51 cannot bind to ssDNA site = failure of HR
What is the process of HR?
dsDNA break is recognized by MRN complex, which collaborates with other proteins to form resected ssDNA and exchange this for RAD51. Arrival of RAD51 is facilitated by XRCC2/3, BRCA1/2, and RAD54B. The RAD51-filament can invade the sister chromatid strand to form a D-loop. DNA polymerases extends the invading strand and the D-loop is resolved
BRCA2 and RAD51 were overexpressed in which canine tumor?
canine mammary carcinomas (primary tumors and nodal metastasis)
What about the mouse telomere suggests that mice do not rely on telomere length to limit the replicative capacity of normal cells?
Mice: The double-stranded region of telomeric DNA is 5X longer (40-50 kb) than in humans, which means mouse telomeres are so long that they are never in danger of eroding down to critically short lengths
- mTERT knockout = conversion of a thoracic vertebra to lumbar vertebra
- mTR mRNA subunit knockout = initially does not change phenotype; mice must reproduce to 5-6 generations before their telomeres are short enough to cause disease
What about the mouse telomere suggests that mice do not rely on telomere length to limit the replicative capacity of normal cells?
Mice: The double-stranded region of telomeric DNA is 5X longer (40-50 kb) than in humans, which means mouse telomeres are so long that they are never in danger of eroding down to critically short lengths
- mTERT knockout = conversion of a thoracic vertebra to lumbar vertebra
- mTR mRNA subunit knockout = initially does not change phenotype; mice must reproduce to 5-6 generations before their telomeres are short enough to cause disease
Which phosphorylates threonine on CDK?
What binds to threonine in cyclin/CDK complexes?
- phosphorylated by the CDK-activating kinase (CAK)
- in humans CAK is CYCLIN H-CDK7 (active throughout cell cycle)
o Causes the activation loop (aka t loop) to move out of the catalytic cleft for phosphorylation
o Phosphorylation AND cyclin binding are required for CDK activation
Answer all of the following regarding the INK4 family:
- what are the members of the family?
- active in which part of the cell cycle?
- what is the main target?
- Includes p16INK4A, p17INK4B, p18INK4C, and p19INK4D
- in early and mid-G1
- binds CDK4 and 6 to prevent their binding to Cyclin D
Answer all of the following regarding the CIP/KIP family family:
- what are the members of the family?
- active in which part of the cell cycle?
- what is the main target?
more generalized inhibitors
- Includes p21CIP1, p27KIP1 and p57KIP2
- P21 and p27 actually promote formation of Cyclin D-CDK4/6 complexes during G1
- inhibit Cyclin E-CDK2, Cyclin A-CDK2, and Cyclin B-CDK1
Which kinase would be specific for M phase inhibition?
o It phosphorylates multiple kinetochore, centrosome, and spindle proteins
Polo-like kinase inhibitor
PLK1 is one of the 4 kinases that govern mitosis (others incl. Cyclin B-CDK1, Aurora A and Aurora B)
What are the kinetochore-associated proteins that are critical constituents of the spindle-checkpoint pathway?
MAD2, BUBR1, BUB1, and BUB3
Match the correct kinetochore-associated protein with it’s particular job: MAD2, BUBR1, BUB1, and BUB3
- regulate mitotic progression by direct inhibition of APC machinery
- mediate mitotic arrest after disruption of microtubules
MAD2 and BUBR1- regulate mitotic progression by direct inhibition of APC machinery
BUB1 and BUB3- mediate mitotic arrest after disruption of microtubules
T/F: Cells that lack BUB1 or BUB3 undergo mitotic arrest when treated with spindle-distupting agents?
FALSE: Cells that lack BUB1 or BUB3 DO NOT undergo mitotic arrest
- What is the main job of RB?
- Describe the phosphorylation process that occurs in each phase of the cell cycle?
- What dephosphorylates RB to prepare for the next cell cycle?
- Rb governs G1/S transition
- G0: unphosphorylated
o Early to mid-G1: hypophosphorylated (weakly phosphorylated) at serine/threonine residues
o After the R point: hyperphosphorylated; remains this way for rest of cell cycle - protein phosphatase type 1 (PP1)
- Phosphorylation of RB is governed by what factors?
2. Describe the phosphorylation process and the interaction of RB with E2Fs in the cell cycle?
- cyclins-CDK
- Early to mid G1: weak phosphorylation by Cyclin D-CDK4/6
o R point: rise in Cyclin E triggers hyperphosphorylation by Cyclin E-CDK2
• Hypo- or unphosphorylated RB binds E2Fs, preventing their activity
• Hyperphosphorylated RB releases E2Fs–> gene transcription to enter S phase
o E2Fs 1, 2 and 3 facilitate gene transcription by binding to histone acetylases and unwinding chromatin
• Factors affecting RB o Oncoproteins (E1A, large T antigen, E7): bind to hypophosphorylated pRb
- Phosphorylation of RB is governed by what factors?
2. Describe the phosphorylation process and the interaction of RB with E2Fs in the cell cycle?
- cyclins-CDK
- Early to mid G1: weak phosphorylation by Cyclin D-CDK4/6
o R point: rise in Cyclin E triggers hyperphosphorylation by Cyclin E-CDK2
• Hypo- or unphosphorylated RB binds E2Fs, preventing their activity
• Hyperphosphorylated RB releases E2Fs–> gene transcription to enter S phase
o E2Fs 1, 2 and 3 facilitate gene transcription by binding to histone acetylases and unwinding chromatin
Which of the E2Fs are inhibitory and during which phases of the cell cycle?
E2F4 and 5 repress transcription during early/mid G1
E2F7 and 8 repress transcription during late G1
Which of the E2Fs are inhibitory and during which phases of the cell cycle?
E2F4 and 5 repress transcription during early/mid G1
E2F7 and 8 repress transcription during late G1
List the million things that can affect RB.
Good luck tyring to remember all of these!
o Oncoproteins (E1A, large T antigen, E7): bind to hypophosphorylated pRb
o Ras –> Raf –> MAPK –> Fos and Jun –>AP-1 –> cyclin D1 transcription
o Ras –>PI3K -> Akt/PKB –> phosphorylate GSK-3B –> B-catenin escapes to nucleus –> Tcf/LEF stimulate cyclin D transcription
o Ras –> Raf –> MEK –> ERK –> phosphorylation of Cyclin D = destruction
o TGF-B –> SMADs migrate to nucleus to induce expression of p15INK4B and p21CIP1
