Replication Flashcards
Rous sarcoma tumor experiment
Cell free infiltrate from sarcoma from one chicken is injected into another. Creates a new sarcoma.
Retroviral life cycle
- DNA copy of genome is made
- DNA integrates randomly into the genome of the host cell
- Viral promoter makes new copies of the RNA genome, which leads to the production of viral particles.
- accidental integration next to cellular gene leads to highjacking of gene.
- expression of truncated gene product by virus can be oncogenic (replication-driving).
- Can also render tumor suppressor gene non-functional
Creation of a conditional mutant today
- Inducible expression of a gene product.
Common targets of viral hijacking/promotion
Src, Raf, Ras, Myc, Jun, Smads, Fos –| p53, CycD,
DNA viral strategy
encode proteins that push quiescent cells to divide, creating a permissive environment for its replication. Like RNA viruses, consequence of infection is proliferative advantage.
Cell division cycle
(G0) -> G1 -> S(ynthesis) -> G2 -> M ->
Cell cycle requirements
- alternation of segregation and chromosome duplication
- Cell must only divide when prompted
- they must have enough nutrients to finish division
- DNA must be fully duplicated
Cell fusion experiments
- G1,S,G2 + M -> M
Replication already committed to. - G1 + S -> S
G1 to S induced with S phase inducer - S + G2 -> S/G2
G2 cells lack S phase inducer
DNA related checkpoints for cell division
- Damaged or missing DNA before mitosis
- Damaged DNA before synthesis
- chromosome attachment to spindle before anaphase
- DNA in intercellular bridge before cytokinesis.
- nuclear pores are reassembled before cytokinesis.
Restriction point
A point in G1 in which cells commit to mitotic fate. Determined using serum restriction experiments. Before R-point -> G0, after, -> M.
Viruses have proteins that circumvent R-point (subvert p53 and RB) to create a permissive environment for replication.
Marks the point when cells pass from being influenced by external signals to being governed by internal signals.
Cell cycle analysis experiments
- Use DNA-binding Dye. FACS sort with more fluorescent dye. 1 -> G1, 1-2 -> S, 2 -> G2, M
- FUCCI method: fluorescent protein-tagged markers for specific cell cycle phases.
RB protein
E2F1 regulates the gene expression program required for S-phase entry. Bound to E2F-DP.
RB binds to E2F-DP repressing the DNA it is bound to.
RB must be inhibited (normally by phosphorylation) to pass the R point.
Part of pocket protein family, w/ p107, p130
Cyclin Dependent Protein Kinases
Group of kinases regulating cell cycle progression, which are dependent on partner cyclins to be activated.
pre-R G1: CDK3/cycC, CDK4/6/cycD post-R G1: CDK2/cycE, S: CDK2/cycA, S, G2: CDK1/cycA M: CDK1/cycB
CDKs also have other roles, ex CDK4 involved in DNA damage response.
Role of Cyclins in R point transition.
Unphosphorylated pRB, inactive E2F-DP.
CycD, CDK4/6 hypophosphorylate pRB. Leads to hyperphosphorylation by cycE/CDK2.
Active E2F1.
Target genes produce more E2F and CycE, leading to switch behavior. Only nuclear Cyclin D is relevant for R point control, cyclin E takes over after R point transition.
DREAM complex
Repressive complex, promoting deacetylaiton of histones and shut down gene expression. p130 acts as glue that keeps complex together, phosphorylation leads to complex disassembly.
Constitutes a separate control mechanism from RB.
Formation of complex started by MuvB phosphorylation. Important for signaling G0 and senescence.
MuvB
part of DREAM complex, first represses gene expression. Activates cell cycle progression genes during G2/M.
Why can’t genes from S. plombe be transcribed by budding yeast?
Budding yeast can’t recognize S. plombe introns. Require very specific intronic sequences.
Regulation of CDKs
- Certain phosphorylation patterns lead to activation or repression.
- CKS allows targeting of genes.
- Cyclins activate, can be ubiquinated and destroyed for degradation.
- CKI binding inhibits.
- nuclear localization.
- temporal expression (linked to cell cycle)
CKI’s
- p57
- p27
- p21
all block CDK1,2 but p21,p27 have double role in temporal regulation by activating CycD/CDK4/6 assembly. CDK2 allowed to act in late G1, because active CDK4 consumes all p21,p27
-p(15-19) block assembly of CycD/CDK4/6
Translocated to cytoplasm when mitogen signaling occurs.
CycD
Regulates R point control, many growth signaling pathways, cytokines, Wnt etc converge on activating CycD.
Only essential cyclin. Others are conditionally essential.
Can be hijacked by viral gene promoter insertion.
Lesson: there is considerable redundancy/promiscuity between cyclins.
G0 and cyclins
Have CDKs but no cyclins, therefore p21, p27 which are present have no effect.
Myc and growth signaling
Acts as dimer with different partners. With Max activates gene expression for CycD/CDK4/6. with Miz-1 is repressor p21,p27, p15. Converges on activating RB through activating E2Fs.
Myc signal shut down through TGF-β receptor which used Smad2 as 2º messenger, oncogenic target.
Targeted Proteolysis by Ubiquitination
Example:
E1+ATP +Ub -> E1-Ub -> E2-Ub -> CycB-Ub
Important for:
- Removal of regulatory molecules
- Quality control, gets rid of denatured proteins
- Opposed by de-ubiquitinylases.
Ubiquitin Ligases
Common features:
- Cullin scaffold
- Adapter protein binds to linker & substrate
- RING protein mediates E2 binding.
Substrates are recognized by phosphorylation or hydroxylation.
Adapter proteins are regulated by periodic expression, and destruction.
Proteolysis and the cell cycle
- Cdc6 locks down origin recognition complex
- Phosphorylation of cdc6 by cdk1 results in ubiquitination of cdc6, and destruction.
- Allows S phase to proceed, and ensures that S phase occurs only once per cycle.
Also destroys E2F.
R-point deregulation in Cancer
- RB inactivated by mutation
- D and E cyclins overexpressed
- Mutation in CDK4, preventing p16 binding.
- deletion of p15 or p16
- Decreased expression of p27
- p53 subverted through mutation, silencing of ARF, or up regulation of mdm2.
- degradation of p27 through cycE-CDK2 phosphorylation.
- over-expression of SKP2.
- loss of phosphorylation/ubi sites of β-cat
- mislocalization of p21, p27 to cytoplasm (aberrant mitogenic signaling)
- elimination of p53, RB by viral proteins
- GF (aberrant myc), infl. (NF-κB) signaling increasing cycD levels
- Smad 2 silencing allowing myc inhibition of p15.
- Inactivaiton of PP2A
p15-19
CKI for cycD-CDK4/6
p21, p27
Activator for cycD-CDK4/6 and CDK for cycE-CDK2.
cycE-CDK2 can also inhibit p27 by phosphorylating it, leading to degradation by SCF-SKP2.
The CDKN2A and CDKN2B loci
encode p15,16, p14 for ARF. Frequently mutated in cancer.
p53
Is an activator of p21 –| cycE. Prevents entry into late G1. Important sensor for genotoxic stress and also regulates entry into senescence/apoptosis/DNA repair.
Frequently mutated, dominant negative phenotype. inhibitor mdm2 often mutated to not be effective. ARF (p14) frequently silenced. ARF inhibits mdm2.
ARF
tumor suppressor downstream of Myc and Ras (sensor for oncogenic signaling), inhibits mdm2. Silenced ARF leads to too much mdm2 signaling, inhibited p53.
Degraded by HPV E6 protein.
β catenin
Oncogene, regulator for cycD transcription.
Deregulation of myc expression
- Retroviral transduction
- Retroviral promoter insertion
- Chromosomal translocation
- Gene amplification
miRNAs
misregulation important in some cancers. Tumor suppressing miRNAs:
1. Genomic loss
2. Loss of histone acetylation (epigenetic silencing)
3. Repression by oncogenic TF
4. Loss of tumor suppressor TF
mostly converge of CDK2/4/6
Oncogenic miRNAs:
Loss of normal repressive epigenetic marks.
Exiting/Avoiding senescence
Acute ablation of RB will allow senescent cells to resume proliferation.
Loss of p16 or p53 will prevent senescence.
PP2A
Opposes CDK. Each forms positive-feedback group and switch behavior.
Phosphatases in PP2A group are pro-interphase. Inactivated in many cancers.
CDK1 –> greatwall –> ENSA –| PP2A
Exit from mitosis
CDK1 phosphorylates (thus activating) ubiquitin ligase (cdc25) for cycB, leading to its own inactivation. Exit from mitotic cycle. Switch swings to phosphatases.
Synthetic lethality in cyclins
Cancers are genetically unstable and my come to rely on 1 or 2 cyclins (redundancy). Screens for synthetic lethality can identify whether these are promising targets for cotherapy.
Concept of dominant negative alleles
The protein is still present but inactive. If overexpressed it may titrate away regulators, interacting proteins or substrates by forming non-productive complexes. Note the difference between this and a null allele, when the gene is deleted or disrupted and no protein is produced.
Large T antigen
Inactivates RB and p53
Small T antigen
Inactivates PP2A, promoting CDK overactivation
Ras
Sends growth signals
