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Replication Flashcards

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Biology 101 flashcards
1
Q

Rous sarcoma tumor experiment

A

Cell free infiltrate from sarcoma from one chicken is injected into another. Creates a new sarcoma.

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2
Q

Retroviral life cycle

A
  • 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
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3
Q

Creation of a conditional mutant today

A
  • Inducible expression of a gene product.
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4
Q

Common targets of viral hijacking/promotion

A

Src, Raf, Ras, Myc, Jun, Smads, Fos –| p53, CycD,

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5
Q

DNA viral strategy

A

encode proteins that push quiescent cells to divide, creating a permissive environment for its replication. Like RNA viruses, consequence of infection is proliferative advantage.

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6
Q

Cell division cycle

A

(G0) -> G1 -> S(ynthesis) -> G2 -> M ->

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7
Q

Cell cycle requirements

A
  • 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
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8
Q

Cell fusion experiments

A
  • 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
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9
Q

DNA related checkpoints for cell division

A
  • 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.
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10
Q

Restriction point

A

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.

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11
Q

Cell cycle analysis experiments

A
  1. Use DNA-binding Dye. FACS sort with more fluorescent dye. 1 -> G1, 1-2 -> S, 2 -> G2, M
  2. FUCCI method: fluorescent protein-tagged markers for specific cell cycle phases.
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12
Q

RB protein

A

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

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13
Q

Cyclin Dependent Protein Kinases

A

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.

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14
Q

Role of Cyclins in R point transition.

A

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.

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15
Q

DREAM complex

A

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.

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16
Q

MuvB

A

part of DREAM complex, first represses gene expression. Activates cell cycle progression genes during G2/M.

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17
Q

Why can’t genes from S. plombe be transcribed by budding yeast?

A

Budding yeast can’t recognize S. plombe introns. Require very specific intronic sequences.

18
Q

Regulation of CDKs

A
  • 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)
19
Q

CKI’s

A
  • 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.

20
Q

CycD

A

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.

21
Q

G0 and cyclins

A

Have CDKs but no cyclins, therefore p21, p27 which are present have no effect.

22
Q

Myc and growth signaling

A

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.

23
Q

Targeted Proteolysis by Ubiquitination

A

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.
24
Q

Ubiquitin Ligases

A

Common features:

  1. Cullin scaffold
  2. Adapter protein binds to linker & substrate
  3. RING protein mediates E2 binding.

Substrates are recognized by phosphorylation or hydroxylation.

Adapter proteins are regulated by periodic expression, and destruction.

25
Q

Proteolysis and the cell cycle

A
  • 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.

26
Q

R-point deregulation in Cancer

A
  • 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
27
Q

p15-19

A

CKI for cycD-CDK4/6

28
Q

p21, p27

A

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.

29
Q

The CDKN2A and CDKN2B loci

A

encode p15,16, p14 for ARF. Frequently mutated in cancer.

30
Q

p53

A

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.

31
Q

ARF

A

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.

32
Q

β catenin

A

Oncogene, regulator for cycD transcription.

33
Q

Deregulation of myc expression

A
  1. Retroviral transduction
  2. Retroviral promoter insertion
  3. Chromosomal translocation
  4. Gene amplification
34
Q

miRNAs

A

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.

35
Q

Exiting/Avoiding senescence

A

Acute ablation of RB will allow senescent cells to resume proliferation.
Loss of p16 or p53 will prevent senescence.

36
Q

PP2A

A

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

37
Q

Exit from mitosis

A

CDK1 phosphorylates (thus activating) ubiquitin ligase (cdc25) for cycB, leading to its own inactivation. Exit from mitotic cycle. Switch swings to phosphatases.

38
Q

Synthetic lethality in cyclins

A

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.

39
Q

Concept of dominant negative alleles

A

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.

40
Q

Large T antigen

A

Inactivates RB and p53

41
Q

Small T antigen

A

Inactivates PP2A, promoting CDK overactivation

42
Q

Ras

A

Sends growth signals

Cancer Bio (7 decks)

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