1.6 Control of the cell cycle pt1

G1 phase

The cell grows and synthesizes all cellular components that are essential for DNA duplication.

S phase

DNA synthesis & replicates the genetic material. Each chromosome is now duplicated and consists of two sister chromatids.

G2 phase

The cell prepares for cell division that occurs in the M phase.

Mitosis (M) phase.

Chromosome segregation (mitosis) followed by cell division (cytokinesis), generates 2 identical daughter cells.

interphase.

The phase of the cell cycle that encompasses G1, S, and G2 phases, during which the cell grows, duplicates its DNA, and prepares for mitosis.

G1 checkpoint

ensures that conditions are favorable for replication. (growth factor signals, DNA integrity, cell size, protein reserves are assessed)

Which checkpoint is often referred to as the restriction point

G1

What happens if the cell does not pass G1 checkpoint

enters a resting state called G0 to await further signals when conditions improve

Which cells remain in G0 for their entire lifetime

neurons, skeletal muscle cells are typically in G0

The transition from G1 to S phase is ruled by CDK4/6

CDK4/6- cyclin D and CDK2-cyclin E complexes.

G2 checkpoint

ensure that all chromosomes have been replicated and that the replicated DNA is not damaged.

If DNA damage is detected during the G2 checkpoint, what happens to the cell

the cell cycle is paused, and the cell will attempt to either complete DNA replication or repair the damaged DNA.

The G2 phase is ruled by which complexes

CDK2-cyclin A and CDK2-cyclin E complexes phosphorylate Forkhead box M1 protein (FoxM1, a transcription factor) and activate the expression of FoxM1 target genes that allow for the transition to M-phase

Role of CDK2-cyclin A and CDK2-cyclin E complexes during G2 phase

phosphorylate Forkhead box M1 protein and activate the expression of FoxM1 target genes that allow for the transition to M-phase

M checkpoint

spindle checkpoint: the attachment of each centromere to the spindle fibers is assessed. Mitosis will only proceed if this is correct.

Mitotic CDKs

CDK1-cyclin A and CDK1-cyclin B complexes.

When mitotic CDKs are high during M phase, factors that initiate DNA replication are

inhibited to prevent replication until the next cell cycle.

Why would mitotic CDKs want to inhibit DNA replication factors

second round of DNA synthesis does not occur until mitosis is complete and the cell has passed the next G1 checkpoint.

Mitotic CDKs activate which complex

anaphase promotion complex (APC/C)

anaphase promotion complex (APC/C)

allow chromatids to separate at anaphase and complete mitosis to decreases the possibility of aneuploidy

Regulation of the cell cycle involves a combination of

specialized cyclin proteins associated with a cyclindependent kinases (CDK)

Cyclins and Cyclin-Dependent Kinases (CDKs)

regulator molecules of the cell cycle that form complexes with each other and are unique to each phase

CDKs

serine/threonine protein kinase enzymes that phosphorylate specific target proteins

Cyclins

regulatory proteins with no catalytic activity that bind to CDKs and activate them

CDKs cannot phosphorylate until what key thing happens

they are bound and activated by a cyclin.

Cyclins/CDKs associated with mitosis

CDK1, cyclin A and B

Cyclins/CDKs associated with G1/S checkpoint and S phase

CDK2 and cyclin A and B

Cyclins/CDKs associated with entry into G1 cycle

CDK4—> cyclin D
CDK6 —> cyclin D

Cyclins and CDKs undergo a constant cycle of

synthesis and degradation during the cell cycle.

Before a cell can progress from one phase of the cell cycle to the next

it must degrade the cyclin that characterizes that phase of the cell cycle to allow for the activation of the next phase's cyclins and CDKs.

Negative regulators

halt the cell cycle primarily at the G1 checkpoint

Cyclin/CDKs are negatively regulated by

cyclin-dependent kinase inhibitors (CKIs).

Examples of negative regulators

Tumor-suppressor proteins,

Tumor-suppressor proteins,

p53, p21, retinoblastoma protein (Rb)

p53

halts the cell cycle if damaged DNA is detected & recruits enzymes to repair the DNA.

p21

enforces the halt dictated by p53 by inhibiting the activity of CDK/cyclin complexes.

Rb

binds to E2F transcription factor and blocks the production of proteins needed for G1/S transition.

E2f

transcription factor important for cell growth which induces S-phase progression in association with CDK2-cyclin E complex

How does Rb interact with E2f

it binds to the factor so the proteins needed for entry into S phase cannot be produced

How is the Rb block on Ef2 removed

phosphorylated by the G1 CDK4/6-cyclin D complex, E2F is released, cell enters into S phase

Ras protein

proto-oncogene that activates G1 checkpoint cyclins

If Ras protein is mutated,

it is constantly active, will constantly activate G1 cyclins, causing uncontrolled cell division and potentially leading to cancer.

Anticancer drugs often target

cell cycle signaling pathways using CDK inhibitors

1st & 2nd gen anticancer drugs

inhibit of a range of CDKs

3rd gen anticancer drugs

specific inhibitors of CDK4 and CDK6 (G1 CDKs)

Side effects of anticancer drugs

Neutropenia and Thrombocytopenia are common adverse effects that result from the targeting of rapidly dividing cells.

all cells in the body have the same

genetic material but express different proteins due to gene regulation

Exome

contains all the exons of RNA that code for proteins in a genome.

Proteome

refers to the entire set of proteins expressed by a genome, cell, tissue, or organism at a given time.

housekeeping genes

are always transcribed
ex: DNA polymerase, metabolism proteins

Examples of specialized genes whose transcription is either on/off

Hb in red blood cells but not in other cells

Finely tuned genes that can change from external signals

starvation leads to the synthesis specialized enzymes in the liver à amino acids to glucose

Many genes are regulated primarily at the level of

transcription

DNA exists as condensed structures called

chromatin or chromosomes

chromatin or chromosomes are formed when

DNA strands wrap around histone, and non-histone proteins

Nucleosome

1st step of DNA compaction, consisting of a segment of DNA wound around a core of histone proteins.

heterochromatin

is a form of densely packed chromatin that is transcriptionally inactive and often found at the periphery of the nucleus.

Euchromatin

is a form of loosely packed chromatin that is transcriptionally active, allowing for gene expression and DNA replication.

DNA mehtylation

adds methyl (-CH3) groups to the DNA using DNA methyltransferase

Where does methylation of DNA occur

on cytosine nucleotides that are found next to a guanine nucleotide and are linked by a phosphate group in the DNA sequence = CpG dinucleotide

DNA methylation forms

5-methyl-cytosine, which projects into the major groove of DNA and inhibits transcription

Histone proteins

pack the DNA into nucleosome complexes.

1 nucleosome is made of

2 each of H2A, H2B, H3, H4 histone proteins

Nucleosomes are further packed together by

histone N-terminal tails
histone H1 molecules

2 mechanisms to change chromatin structure:

1. enzymatic modification of the histone N-terminal tails.
   2. ATP-driven chromatin remodeling complexes

enzymatic modification of the histone N-terminal tails.

o acetylation
o methylation
o phosphorylation

What enzyme mediates acetylation of histone N terminal tails

histone acetyl transferase (HAT) enzyme

What enzyme mediates methylation of histone N terminal tails

histone methyl transferase (HMT) enzyme

What enzyme mediates removal of acetyl groups from histone N terminal tails

histone deacetylase complex transferase (HDAC)

the “histone code”

The pattern of histone modifications

histone methylation promotes formation of

heterochromatin and transcriptional repression.

Acetylation of histones promotes formation of

euchromatin and transcriptional activation.

ATP-driven chromatin remodeling complex

is thought to “push” on the DNA and “loosen” the attachment to the histone core
can also add/remove/change nucleosome proteins

Coding region of a eukaryotic gene is flanked by

5’ and 3’ UTR

Promoter.

A region of DNA upstream from a gene which is the binding site for transcription factors (TFs) and the transcriptional apparatus, RNA polymerase, etc

Regulatory sequence

Binding sites on DNA for a variety of cell-specific or tissue-specific TFs. Can be located at a distance from the gene they regulate

Genetic switches

Gene transcription can be turned on and off in response to a variety of signals.

Components of genetic switches

1. specific DNA sequences
2. proteins that bind to these DNA sequences

DNA binding proteins

are also called gene regulatory proteins
also called transcription factors
Function with gene regulation

A consensus sequence

a conserved nucleotide sequence of DNA, RNA, or amino acid sequence that is generally used for molecular interactions.

Transcription factors recognize

SPECIFIC DNA sequences

Structural motifs of transcription factors

zinc fingers or leucine zippers, helix-turn-helix that recognize and target precise DNA sequences.

p53 is a is a well-known

master transcription factor and a critical tumor suppressor protein that uses protruding peptide loops to target precise DNA sequences.

Mutations of the p53 gene is regarded as a hallmark of

cancer cells

What part of the DNA helix is actually read and recognized by transcription factors

the outside of the helix so it doesn’t have to be unwound

When TFs bind to DNA and turn gene transcription on

positive control

TFs that mediate positive control (turn on) are called

activators or gene activator proteins.

when TFs bind to DNA and turn gene transcription off

negative control

TFs that mediate negative control (turn off) are called

repressors or gene repressor proteins.

A single type of TF can regulate

the expression of different genes and can be involved in both positive and negative control of transcription.

TFs can form

homomeric and heteromeric proteins (Dimers, trimers, tetramers etc.)

Why would we want TFs to be able to make homomeric/heteromeric proteins

This expands the repertoire of DNA sequences that gene regulatory proteins can recognize.

TFs can assemble as

complexes on DNA

Combinatorial Gene Regulation

During development different cell types and different tissues are created due to different combinations of TFs

TFs play a major role in the differentiation of stem cells to

teeth

Developmental defects in teeth usually occur from

mutations in TFs

Mutations in PAX9

results in partial or total anadontia.

Mutations in the RUNX2

causes supernumerary teeth

The TATA box

a common promoter DNA sequence in eukaryotes where the TATA binding protein can bind

Before transcription can begin, RNA polymerase II requires

general transcription factors to assemble at the promoter
ex: TBP (TATA Binding Protein), TFIIA, TFIIB, TFIID