The Eukaryotic Cell Cycle, Part 2

Lecture 24

Mitotic CDKS induce entry into mitosis in al eukaryotes by

inducing chromosome condensation, nuclear envelope breakdown, and spindle formation

Mitotic CDKs are inactivated by

inhibitory phosphorlyation of the CDK subunit until completion of DNA replication 

mitotic CDKs promote their own activation through 

positive feedback loops that inactivate Wee1 kinase and activate Cdc25 phosphatase 

the kinetochore on each compacted sister chromatid attaches 

to microtubules emanating from opposite spindle poles 

cells ensure bi-orientatoin of sister chromatids in the spindle by

a tension-based mechanism

mitotic cyclin synthesized during

S and G2 activare mitotic CDKS

the tyrosine and threonine residues must be 

dephosphorlyated for the CDK to become active 

the degradation of mitotic cyclin at the completion of mitosis allows

the daughter cells to enter the G1 phase

dynamically assembling-disassembling microtubules anchored by their minus (_) ends to

each spindle pole search-and-capture chromosomes 

chromosome motor proteins propel chromosomes to the 

plus (+) end of microtubules, where each of the two sister chromatid kinetochore attaches to several microtubules from opposite poles 

the outer kinetochore Dam1 complex and part of the Ndt80 complex form

a ring around a captured spindle microtubule near its plus end

inner kinetochore protein complexes link the

inner kinetochore to the outer kinetochore and to the chromosome centromere

Aurora B kinase detects and destabilizes

incorrect attachments at the microtubule-kinetochore interface

tension generated by ampitelic attachment pulls

sister kinetochores apart, thus disrupting the interaction of Aurora B at the innermost portion of the centromere with microtubule binding sites on the outermost centromere 

merotelic attachment -

one kinetochore attaches to microtubules emanating from two opposite spindle poles 

syntelic attachment - 

both sister kinetochores attach to microtubules emanating from the same spindle pole 

monotelic attachment -

only one of the two sister kinetochores attaches to microtubules

metaphase chromosome - chromosome condensation (and untangling) into

travel-friendly structures facilitates chromosomes organization

metaphase chromosome - both sister chromatids

are fully condensed

linear compaction:

the folding of chromosomes into consecutive loops by condensing proteins (related to cohesions) leads to the formation of proteinaceous fibers at loop bases

condensin helps

compaction of intramolecular DNA

cohesin and codensin both contain

SMC proteins (structural maintenance of chromosomes) that fold back on themselves to help compact chromatin

axial compaction:

condensin generates further compression into highly condensed chromosomes 

cohesion cleavage by separate initiates 

chromosome segregation during anaphase 

exit from mitosis is triggered by mitotic cylindrical degradation and requires

protein phosphatase reversal of mitotic CDK phosphorylation of many different proteins, permitting mitotic spindle disassembly, decondensation of chromosomes, and reassembly of the nuclear envelope 

cytokinesis position is coordinated with

spindle position

S phase to M metaphase:

• sister chromatids are held together by cohesins

• separase protease activity is inhibited by CDK phosphorlyation and securing binding 

when spindle checkpoints controls indicate all kinetochores are attached to microtubules and the spindle is properly assembled and orientated, the cdc20 specificity factor directs 

the anaphase promoting complex (APC) to ubiquitylate securing and mitotic cyclin, targeting them for proteasome degradation 

securing degradation and dephosphorlyatoin releases separase to proteolyses Scc1, breaking the 

cohesin attachments of the sister chromatids and initiating anaphase during which the disconnected sister chromatids are pulled toward opposite spindle poles 

The spindle checkpoint pathway ensures

each kinetochore is properly attached to spindle microtubules before separation of duplicated chromosomes after anaphase initiation

The proteins responsible for the SAC signal compose the mitotic checkpoint complex which include

MAD2/MAD3 (mitotic arrest deficient), BUB3 (budding uninhibited by benzimidazole), Bub1 kinase, and CDC20

the spindle assembly checkpoint steps the cell cycle by negatively regulating

CDC20, thereby preventing the activation of the polyubiquitlyation activates of the anaphase promoting complex 

the checkpoint kinase Mps1 phosphorylated the

outer kinetochore component Knl1 of unattached kinetochores

Phosphorylated Knl1 binds the checkpoint kinase Bub1-Bub3 and the checkpoint protein Mad3, which recruits

the Mad1-Mad2 complex to the kinetochore and activates Mad2 (Mad2-A) activity

Mad2-A binds APC/C^Cdc20  and completely prevents it from recognizing and ubiquitinylating its substrates by recruiting

the mitotic checkpoint complex (MCC).

When microtubules attach and generate tension on the kinetochore, protein phosphatase 1 eliminates checkpoint protein binding sites by

dephosphorylating Knl1, and the MCC disassembles, activating APC to ubiquitinylate substrate proteins for mitosis to proceed. 

inactivation of mitotic CDKs:

• mitotic cyclin destruction targeted by APC

• accumulation of the CDK inhibitor Sic1, which inhibits S phase CDKs until cells enter the cell cycle 

mitosis - mitotic CDK activity inhibits

its APC/C^Cdh1 and Sic1 inhibitors

the protein phosphatase Cdc14 triggers

exits from mitosis

Exit from mitosis − The mitotic exit network (an anaphase spindle position-sensitive GTP signaling pathway)

activates Cdc14 protein phosphatase

Cdc14 protein phosphatase:

•Activates APC/C^Cdh1 by dephosphorylating it

•Promotes Sic1 accumulation

•Dephosphorylates the many mitotic CDK substrates

Cyclin-dependent kinases and ubiquitin-mediated protein degradation ensure that

a cell cycle stage is not initiated until the previous one has been completed and that each cell cycle step occurs accurately.

checkpoint pathways ensure

the next cell cycle event does not occur prior to the completion of the preceding one

checkpoint pathways are compromised of

event sensors, a signaling pathway, and an effector that halts cell cycle progression and activate repair pathways when necessary

checkpoint pathways monitor and respond to

DNA replication and damage, and spindle assembly and position

the highly conserved Hippo signal pathway coordinates

fungi chromosome segregation with mitosis exit and cytokinesis but coordinates metazoan tissue growth with tissue organization

cell capable of stopping

cell cycle at checkpoint

surveillance system at each checkpoint

• damage to chromosomal DNA

• DNA replication incomplete

• chromosomal alignment (M phase) incomplete

passage to next phase inhibited if

requirements not met

cell proliferation controlled by signals

• influence or regulate checkpoints

• specific combination required for cells to multiply 

cancer - result of

mutations that release cells from proliferation and survival controls

cancer ability

• acquire ability to divide without usual restraints

• ability inherited by progeny

• genes = proto-oncogenes & tumor suppressor genes 

antiproliferation genes

• encode proteins involved in cell cycle checkpoints

• cells released from normal division controls by defect in gene

• defect in both copies of gene necessary to produce effect in diploid cell

• termed tumor suppressor genes (p53 gene)

proliferation genes - 

encode proteins that promote cell division 

proliferation genes - include

• proteins in signaling pathways for growth factors or proteins that regulate cell survival or apoptosis

• proto-oncogenes

  • defective gene = oncogene

defect in one copy of gene necessary to produce

effect in diploid cell

proliferation genes - types of proteins abnormalities 

• produced by cell that doesn’t normally produce it 

• produced in excessive amounts

• incorrect folding leading to uncontrolled activity 

checkpoint pathway surveillance mechanisms that monitor cell cycle conditions including DNA replication and damage ensure that

the next cell cycle event is not initiated until the previous one has been completed 

ATR kinase: 

• activation leads to arrest in G2 phase

• activated by DNA lesions other than strand breaks 

ATM kinase:

• activation leads to arrest in G1 phase

• activated by DNA strand breakage 

active ATM and ATR activate Chk1 and Chk2 protein kinases, which:

• induce DNA repair machinery

• cause cell cycle arrest by inhibiting Cdc25

in metazoan cells, activate the tumor suppressor transcription factor p53, which

• induces cell cycle arrest by activating transcription of rheumatoid arthritis CKI p21 (cyclin-dependent kinase inhibitor) 

• or when DNA damage is severe, activates apoptosis 

DNA damage stimulates the

p53-p21 pathway to inhibit G1 and mitotic CDKs

ATM or ATR (ATM/R) protein kinases

• Stimulate Chk1/2 protein kinases to inhibit Cdc25, which blocks S phase and Mitotic CDKs.

• Activate p53, which induces synthesis of the CKI p21

Replication stress (slow DNA replication fork movement or DNA replication fork collapse) stimulates

the ATR-Chk1 protein kinase cascade to phosphorylate and inactivate Cdc25C, preventing the activation of mitotic CDKs and inhibiting entry into mitosis

DNA damage can halt

proliferation and hi lights a critical tumor suppressor

DNA damage, especially that induced by UV irradiation,

activates an important cell cycle regulator

if a cell senes damaged DNA, it will

halt the cell cycle at the G1-S transition to provide time to repair the damage

surveillance proteins recognize damaged DNA and phosphorylate a

protein called p53

p53 is a timor suppressor gene; so important is the

p53 protein that it is called the “Guardian of the Genome”

p53 is a transcriptional regulator; it

activate transcription of G1 CDK inhibitor called p21

active p21 binds and inhibits the

G1/S CDK and the S-CDK - blocking progression

p53 is one of the most frequently altered gene in

human cancers

meiosis involves

one cycle of chromosome replication followed by two cycles of cell division to produce haploid germ cells

meiosis-specific gene products and activates modulate

the mitotic cell division program to preform meiosis

in diploid eukaryote meiosis, two consecutive chromosome segregation phases generate 

haploid germ cells (eggs and sperms), which can fuse to generate a diploid zygote that develops into a new individual 

Pre-meiotic cells:

contain two copies of each chromosome (2n), one from each parent (only one chromosome from each parent is diagrammed)

during the mitosis, the chromatids of each chromosome

are split apart and separate into two daughter nuclei in a single division

during meiosis, chromosome number is

halved and four daughter haploid cells are formed

meiosis first division -

homologous chromosome pair and then segregate ensuring that daughter cells receive a full haploid set of chromosomes 

meiosis second division - 

the two chromatids are separated

DNA is replicated prior to

meiosis

prophase I consists of several stages:

• chromosomal condensation starts 

• homologous chromosome pair 

synapsis, and it is when

homologues associate via the synaponemal complex (a ladder-like protein complex that forms between homologous chromosomes during meiosis)

the synaptonemal complex allows

interacting chromatids to complete crossing over

synapsed chromosomes forms a

bivalent or tetrad

meiosis increases genetic variability by

mxing maternal and paternal alleles between homologous chromosomes

recombination occurs by the

physical breakage of and ligation of individual DNA molecules

recombination occurs without

the addition or loss of a single base pair

• DNA repair enzymes fill the gaps that develop during the exchange process 

prior to recombination,

DNA strands are aligned by homology search, in which homologous DNA molecules associate with one another

homology search process

• breaks are introduced into one strand of each duplex at corresponding sites

• the gap is subsequently widened

• the two duplexes are joined to each other by Holliday junctions (pairs of DNA crossovers)

Holliday junctions

pairs of DNA crossovers

Prophase I

• synapsis ends

• the synaptonemal complex disappears and homologous chromosomes  start moving apart 

chiasmata are the remaining point of attachment

between homologous chromosomes

• occur where crossing over took place 

recombination chiasmata and a meiosis-specific cohesin subunit are necessary for

specialized meiosis I and chromosome segregation

kinetochores of maternal and paternal sister chromatids attach to

spindle microtubules from opposite spindle poles

maternal and paternal chromosomes are attached to each other by:

recombination chiasmata that result from crossing over between chromatids

cohesins distal to the crossover point, which are cleaved by 

separate during meiotic anaphase I

metaphase I - 

the two homologous chromosomes are aligned at the metaphase plate

metaphase I - process

• both chromatids of one chromosome face the same pole 

• homologous chromosomes are held by one or several chiasmata

• absence of a chiasma can lead to abnormal segregation of chromosomes