Cell Biology - The Cell Cycle

Front 1

What is the Cell cycle?

Back 1

Interphase + Mitosis + Cytokinesis

Interphase:
G1:
-transition from M to S phase
-synthesis of proteins that be required during S phase
-G0 = cells that become arrested in G1 (quiescence)
-check for cell size, nutrients, growth factors (D-type cyclins), and DNA damage
S:
-synthesis phase
-DNA synthesis: double amount of DNA
G2:
-transition from S to M
-synthesis of proteins need in M
-check for cell size and DNA damage

Mitosis:
-Prophase
-Metaphase
-Anaphase
-Telophase
-chromosome segregation, cytoplasmic division
-check for chromosome attachment to spindle

Cytokinesis:
-cytoplasmic divisions after mitosis
-SEPARATE than mitosis + interphase

CDKs:
-cyclin dependant kinases
-specific enzymes present during G1, S, G2, M
-activated kinase adds phosphate to proteins

CKI:
-cyclin dependant kinase inhibitors
-blocks action of kinases

Front 2

What are the association of the different CDKs?

Back 2

G1:
-Cyclin D with CDk4
-Cyclin D with Cdk6

S:
-Cyclin E with CDk2

G2:
-Cyclin A with CDk2

M:
-Cyclin B with Cde2

Front 3

What happens in the G1 phase?

Back 3

1. growth phase and prep time for DNA synthesis of S phase

2. RNA and protein synthesis

3. organelles and intracellular structures are duplicated and cell grows during this phase

4. LENGTH OF G1 MOST VARIABLE

5. rapidly dividing cells such as embryonic cells spend little time in G1

6. Mature cells remain permanently in G1

7. cells not committed to DNA synthesis = G0 phase

8. Restriction point in G

9. If passed through restriction can go into S phase

Front 4

What happens in the S Phase?

Back 4

1. DNA replication occurs

2. 46 chromosomes copied forming sister chromatids

3. DNA helicase (ATP dependant) unwinds DNA

4. Mulitiple replication forks activated on each chromosome

5. entire genome duplicated during time span of S phase

6. Chromosome strands go into tightly coiled heterochromatin when DNA synthesis complete

7. Constant completion time among cell types

8. Actively dividng spend 6 hrs in S phase

Front 5

What happens in the G2 Phase?

Back 5

1. S to M

2. Prep time for nuclear division of mitosis

3. safety gap makes sure DNA synthesis complete before M

4. checkpoint for nuclear integrity

5. lasts 4 hrs

Front 6

What are Aurora Kinases?

Back 6

-discovered in eggs of toad
1. family of serine/threonine kinases
2. play important functional role during mitosis
3. control chromatid segregation

Aurora A:
-functions in prophase
-critical for proper formation of mitotic spindle
-recruitment of proteins to stabilize centrosomal microtubules

Aurora B:
-attaches mitotic spindle to centromere
-cleavage furrow formation during cytokinesis

Aurora C:
-expressed in germ line cells, unclear function

Elevated A B C = human tumors

Inihibition of aurora kinases being investigated as anticancer therapies

Front 7

What happens in the different phases of Mitosis?

Back 7

Prophase:
-condensed chromosomes start to become visible
-centrosomes move apart and nucleolus starts disappearing

Prometaphase:
-nuclear membrane breaks down
-kinetochores accessible to microtubules

Metaphase:
-chromosomes maximally condensed and align at metaphase plate

Anaphase:
-2 sister chromatids separate and move to opposite poles

Telophase:
-nucleus reassembly occurs, cytokinesis occurs

Front 8

What is the Karotype test?

Back 8

test to identify and evaluate size, shape, and number of chromosomes in a sample

-extra missing or abnormal positions can be detected

-samples arranged in prometaphase stage

Front 9

What is responsible for chromosome movement during mitosis and how?

Back 9

Mitotic spindle is responsible

spindle assembly and chromosome attachment:
-kinetochores - located on opposite sides of a chromosome and anchor kinetochore microtubules
-centromere - ensures delivery of one copy to each daughter
-unattached kinetochores - signal for mitotic checkpoint, arrest mitosis until all kinetochores correctly attached to spindle microtubules; preventing loss of a chromosome (aneuploidy)

Front 10

What does Cytokinesis do?

Back 10

divides the Cytoplasm

-driven by actin and myosin

-large organelles (ER, golgi) fragment into smaller vesicles in mitosis and reassemble in daughter cells

Front 11

What provides evidence for control molecules in the cell cycle? What are these control molecules and how were they identified?

Back 11

Cell fusion experiments provide evidence

Mitosis-promoting factor (MPF):
-moves cells through G2 checkpoint
-phosphorylates lamin to break up nuclear membrane
-phosphorylates condensin which triggers chromosome condensation
-mitotic spindle formation
-targeted protein degradation
-during development of a frog oocyte, cell cycle arrested in G2 until hormone stimulates meiosis



Identified by:
cell cycle mutants:
-temperature sensitive
-PEG
-used to fuse cell

Front 12

How is Mitotic Cdk-cyclin Complex activated?

Back 12

1. MPF is inactive

2. An inhibiting kinase add 2 phosphate group to the complex

3. An activating kinase phosphorylates a third site on the complex

4. A phosphatase removes the inhibiting phosphate groups, converting complex into a single phosphorylated form = active MPF

5. Active MPF --> more active MPF

6. Activation of MPF is autocatytic

Front 13

Who won a nobel prize for discoveries of key regulators of cell cycle and who discovered that MPF is autocatalytic?

Back 13

MPF is autocatalytic = Dr. Masui

Nobel prize = Hunt, Hartwell, and Nurse

Front 14

What happens in Dissolution of the Nuclear Lamina, Breakdown of the Nuclear Membrane, and Re-formation of the nuclear envelope?

Back 14

Dissolution of nuclear lamina:
-at mitosis Cdc2 and other protein kinases phosphorylate lamins, causing filaments to dissociate into free lamin dimers

Breakdown of nuclear membrane:
-nuclear membrane fragments into vesicles
-B-type lamins remain bound to these vesicles, while lamins A and C released as free dimers

Re-formation of nuclear envelope:
-binding of membrane vesicles to chromosomes, mediated by integral membrane proteins and B-type lamins
-vesicles fuse, nuclear lamina reassembles, chromosomes decondense

Front 15

How is the G1 restriction point controlled?

Back 15

G1 Cdk-cyclin complex does this by phosphorylating Rb protein (pRb):
-unphosphorylated pRb interacts with transcription factor E2F
-this prevents E2F from stimulating transcription
-G1 Cdk-cyclin phosphorylates pRb causing it to be released from E2F and allowing E2F to stimulate transcription (can be altered by p53)
-once cell crosses restriction point, entry into S phase is ensured

pRb = retinoblastoma protein (tumor suppressor protein)

E2F = family of transcription factors involved in cell cycle regulation and DNA synthesis in mammalian cells

Front 16

How is the spindle assembly checkpoint controlled?

Back 16

Mitotic Cdk-cyclin complex (MPF) activates anaphase-promoting complex:
-anaphase-promoting complex is a ubiqutin ligase that marks proteins for destruction by proteosomes
-triggers breakdown of securin which normally binds and inhibits separase
-when separase is released it targets and cleaves cohesins which causes sister chromatids to separate from each other
-breaks down MPF cyclin to end mitosis
-APC is not active until all chromatids are linked to spindle, actively controlled by kinetochores which bind Mad and Bub proteins when kinetochores unattached
-Mad and Bub inhibit APC by controlling Cdc20 preventing anaphase

Front 17

What are Ubiquitin and Proteosomes?

Back 17

Ubiquitin:
-target proteins for degradation of proteasomes
-substrate recognition protein E3 recognize N-terminal sequence of their targets but exception are degrons (allow proteins to be ubiquitin marked)
-Anaphase promoting complex functions as E3 AND binds proteins containing degrons

Proteosome:
-predominant protease in cytosol

Front 18

What are the 3 major regulatory pathways?

Back 18

1. Spindle Checkpoint:
-Mad and Bubs control Cdc20 and thus control APC

2. DNA replication checkpoint:
-G2 to M is blocked until DNA duplicated
-final dephosphorylation step activating MPF is blocked until DNA replcated

3. DNA damage checkpoint:
-can halt cell cycle at G1, S, or G2
-p53 activated by ATM-PK
-p21 and PUMA (p53 Upregulated Modulator of Apoptosis) up-regulated
-Mdm2 (Murine Double Minute) involved in degradation of p53
-apoptosis if too much damage

Front 19

What are the different growth factors?

Back 19

Stimulatory growth factors:
Activate Ras Pathway:
-Ras mutations found in pancreatic, colon, lung, and bladder cancers (25-30 % of cancers)
Activate PI3 kinase-Akt pathway:
-PTEN mutation found in 50 % prostate cancer, 35 % uterine cancer

Inhibitory growth factors:
-act through Cdk inhibitors
-TGF beta = causes increase in Cdk inhibitor p15 and p21
-p21 plays a major role in preventing damaged DNA from passing G1 checkpoint

Front 20

What is the PI3-kinase Akt Pathway?

Back 20

1. activation of PI 3-kinase by phosphorylated tyrosines found in receptors that have been stimulated by growth factor

2. Activated PI 3-kinase adds a phosphate to PIP2 --> PIP3

3. PIP3 recruits protein kinases to inner surface of plasma membrane leading to phosphorylation and activation of protein kinase Akt

4. Akt suppresses apoptosis and inhibits cell-cycle arrest, promoting cell survival and proliferation

5. pathway is inhibited by PTEN which catalyzes PIP3 --> PIP2

Front 21

What is Equilibrium Density Centrifugation?

Back 21

-showed that DNA replication is semiconservative

-technique was used to distinguish between N15 DNA (Heavy) and N14 DNA (light)

-the density that came up was in between the two DNA strands showing that DNA is semiconservative

Front 22

How is DNA replication in circular DNA?

Back 22

it has bidirectional replication forks from a single origin

this theta replication occurs in bacteria, mitochondria, chloroplasts, and some viruses

Front 23

How is DNA replication in Eukaryotes?

Back 23

involves many replicons

replicons - units of replication of linear DNA

replication starts at Autonomously Replicating Sequence (ARS) which is rich in AT

Front 24

What is the pre-replication complex?

Back 24

Origin Recognition Complex (ORC) binds to replication origin first

MCM complex, helicase, and primase complex bind next (require assistance from helicase loaders)

Pre-replication complex needs polymerase to be completed

Phosphorylation of MCMs initiates replication

Front 25

What are the main type of Eukaryotic and Prokaryotic DNA Polymerases?

Back 25

Prokaryotic:
-I, II, II, IV, V
-DNA polymerase II is the primary one

Eukaryotic:
-a. d. e
-g is found ONLY in mitochondria
-Polymerase a is involved in initial synthesis of DNA strands off RNA primers
-Polymerase d(lagging) and e(leading) primary polymerases, one for each strand

Front 26

What are all the DNA replication proteins in Prokaryotes?

Back 26

DNA Polymerase I:
-proofreading, removes and replaces RNA primers

DNA Polymerase III:
-synthesis of both DNA strands
-main one

DNA Gyrase:
-Type II DNA topoisomerase which relaxes supercoiling

Front 27

What are all the DNA replication proteins in Eukaryotes?

Back 27

DNA polymerase alpha:
-complexes with primase and begins DNA synthesis

DNA Polymerase gamma:
-mitochondrial DNA synthesis

DNA Polymerase delta and epsilon:
-lagging and leading strand synthesis (also DNA repair)

Telomerase:
-uses integral RNA molecule as template and synthesizes DNA for extension

Front 28

What are the DNA replication proteins in both Eukaryotes and Prokaryotes?

Back 28

Primase:
-RNA synthesis

DNA Helicase:
-unwinds double stranded DNA
-requires ATP and breaks H bonds

Single-stranded DNA binding protein (SSB):
-binds to single stranded DNA

DNA Topoisomerase Type I or II:
-Type I makes single stranded cut
-Type II makes double stranded cut
-relaxes DNA supercoiling

DNA Ligase:
-joins together adjacent DNA strands

Initiator proteins:
-binds to origin of replication and begins unwinding of DNA double helix

Front 29

What does DNA synthesis require?

Back 29

1. DNA polymerase requires a RNA PRIMER for DNA synthesis

2. Primer is a small piece of RNA, synthesized by DNA primase (10 base pairs, in bacteria = primosome, in eukaryotes = DNA Polymerase alpha)

3. Once a small piece of RNA is synthesized, DNA polymerase III will begin to add deoxyribonucleotides to the end of the RNA

4. Later during replication, DNA Polymerase I will come along and remove the RNA, replacing the RNA bases with deoxyribonucleotides

Front 30

What are Okasaki Fragments?

Back 30

For lagging strand DNA must be read in the 3' to 5' direction

Since DNA Polymerase can only synthesize DNA in the 5' to 3' direction the DNA must be synthesized in short fragments

These short fragments are Okazaki fragmnents

DNA ligase seals these okazaki fragments together

Front 31

Why is Telomerase so important?

Back 31

Telomeres are at the end of LINEAR DNA and their segments are too small to work on

Telomerase is a protein and an RNA, the RNA has a complementary sequence to the telomere and the protein has polymerase activity so it fills the gap and adds bends and BPs to form a loop and protect the DNA from being damaged

Telomerase also has a capping protein thus one strand is always longer than the other

found in germ and tumor cells but not differentiated cells

Front 32

What is an Oncogene and an example?

Back 32

Oncogene:
-modified gene that codes for a protein and is believed to cause cancer
-genetic mutations resulting in the activation of oncogenes increase the chance that a normal cell will develop into a tumor cell

proto-oncogene = normal gene that can become an oncogene due to mutations

Telomerase is an example of an oncogene:
-cell loses its ability to divide because telomeres are not added to the end of chromosomes

Front 33

Who won the Nobel prize for discovery of chromosome protection by telomeres?

Back 33

Blackburn, Greider, and Szostak

Front 34

What does DNA licensing mean?

Back 34

makes sure DNA is replicated only once

Regulation:
-Cdk blocks re-licensing by phosphorylating and inactivating ORC, MCM, and helicase loaders
-MCM complex helps initiate DNA replication and is displaced in the process
-Geminin blocks binding of MCM complex to DNA

Front 35

What is Blood Syndrome?

Back 35

DNA Helicase is defective causing problem in DNA replication

Front 36

What are drugs that target bacterial replication?

Back 36

Quinolone:
-target bacterial enzyme gyrase (equivalent to mammalian topisomerases) which break the phosphodiester backbone to relieve torsional strain as DNA helix unwinds to allow replication
-quinolone blocks this and inhibits bacterial DNA replication
-does not affect eukaryotes because topoisomerases are not affected

Front 37

What is A-amanitin?

Back 37

a toxin that is a RNA polymerase II inhibitor

blocks transcription of single-copy gene

a-amanitin has no effect on telomerase or any type of DNA polymerase

Front 38

What are the types of DNA damage and how are they repaired?

Back 38

1.
Damage = Nicks in DNA strand
Repair = DNA ligase

2.
Damage = Mismatched bases
Repair = excision and resynthesis

3.
Damage = Uracil in DNA
Repair = removed by DNA uracil glycosylase

4.
Damage = Apurinic or apyrimidinc site
Repair = AP endonuclease

5.
Damage = Pyrimidine dimers (UV)
Repair = enzymatically reversed

6.
Damage = region of DNA
Repair = Excision repair

Front 39

What are ways to repair abnormal nucleotides?

Back 39

Translesional synthesis:
-repair DURING REPLICATION
-DNA polymerase eta can catalyze DNA synthesis across regions with thymine dimers

Excision repair pathways: POST REPLICATION
-Base excision = repair single damaged bases
-Nucleotide Excision Repair (NER): endonuclease
-pathway that corrects thymine dimers (Xeroderma pigmentosum - incapacitated NER mechanism, cannot tolerate sunlight)

Mismatch Repair:
-corrects mutations that involve noncomplementary base pairs
-Hereditary nonpolyposis colon cancer (HNPCC)

Front 40

What are Mutation hot spots and what is a disease associated with it?

Back 40

30 % of point mutations involve C-->T transitions

CpG islands represent a true hot spot because cytosine is frequently methylated at position 5

Rett Syndrome:
-X linked dominant, only females
-mutation in gene encoding methyl-CpG-binding protein 2 (MECP2)