The Cell Cycle, DNA Replication, and Mitosis

Becker’s Chapter 19

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 The Cell Cycle, DNA Replication, and Mitosis

- Growth and reproduce: Fundamental properties of living organisms.

- Cell growth: more proteins, lipids, carbohydrates, nucleic acids…..
- Over-crowding, plasma membrane expand to increase cell size.
- Reduce surface area to volume ratio, reduce capacity for effective exchange (of
gases, nutrients, wastes….) with the environment.
- Cell growth is usually accompanied by cell division to generate 2 identical cells.
- Cell growth increases the number of cells and increases the size of multicellular
organisms, and replaces cells that have died.

- Q: which cell types in human are being replaced rapidly?

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 The Cell Cycle, DNA Replication, and Mitosis
• Cell growth is generally accompanied by cell division, whereby one cell gives
rise to two new daughter cells.

• All the genetic information in the nucleus must be accurately duplicated and
carefully distributed to the daughter cells

• In doing this a cell passes through a series of stages known as the cell cycle.

Overview of the Cell Cycle

• The cell cycle begins when two new cells are formed by division of a parent
cell and ends when one of these cells divides again

• M phase is when the cells actually divide; the nucleus first, followed by the
cytoplasm. Nuclear division is mitosis and division of the cytoplasm is
cytokinesis

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Under microscope, most dramatic event in cell cycle is the M phase.

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 Mitosis is a relatively short part of the cell cycle

• Cells spend very little time in M phase

• Most of the time is spent in interphase, which is composed of

G1 phase, S phase (when DNA is replicated), and G2 phase

• The overall length of the cell cycle is called the generation

time; in cultured mammalian cells this is about 18–24 hours

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 The G phases (The Gap Phases)
• G1 is quite variable depending on cell type;
G2 is shorter and less variable

• During G1 a major decision is made,

whether a cell will divide again; cells that
arrest in G1, waiting for a signal to divide,
are said to be in G0

• Cells that exit the cell cycle are said to

undergo terminal differentiation. Cells at
this stage will not be divided again. Eg,
nerve cells.

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DNA Replication/Synthesis (S Phase)

•The underlying mechanism depends on the

double-helical structure of DNA

•One strand of every new DNA molecule is derived

from the parent molecule

•and the other is new: semi-conservative

replication

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 DNA Polymerases Catalyze the Elongation of DNA Chains
• DNA polymerase is an enzyme that can
copy DNA molecules

• Incoming nucleotides are added to the 3¢

end of the growing DNA chain, so
elongation occurs in the 5¢ to 3¢ direction.

• Nucleotides are linked together by

phosphodiester bond – between a
phosphate group and two 5’ carbon ring
carbohydrates over two ester bonds.

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 DNA Replication Involves Multiple Replicons
• replication is initiated at multiple sites,
creating replication units called replicons.

• At the center of each replicon is a DNA

sequence called an origin of replication,
where DNA synthesis is initiated.

• A multisubunit protein complex known as

the origin recognition complex (ORC) binds
to the replication origin, followed by the
helicase loaders and minichromosome
maintenance (MCM) proteins.

• MCM proteins include DNA helicases that

facilitate DNA replication by unwinding
double helix.

• All proteins together: Pre-replication

complex – and the DNA is said to be
“licensed” for replication.
• After replication begins, replication bubbles
grow

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 DNA synthesis requires RNA primers
• DNA synthesis is initiated by the
formation of short RNA primers

• These are synthesized by primase using a

single DNA strand as the template

• DNA polymerase can only add

nucleotides to the 3¢ end of an existing
nucleotide chain

• Cells contain an enzyme called primase

that synthesizes short (~10 nt) chains of
RNA using DNA as a template. Primase is
able to initiate RNA strands without a
pre-existing chain to add to

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DNA Is Synthesized as Discontinuous Segments That Are
Joined Together by DNA Ligase
• DNA is synthesized in the 5¢ to 3¢ direction, but the two strands of the double
helix are oriented in opposite directions

• One strand (the lagging strand) is synthesized in discontinuous fragments

called Okazaki fragments – need many primers

• The other (the leading strand) is synthesized in a continuous chain.

• These are then joined by DNA ligase to form a continuous new 3¢ to 5¢ DNA
strand

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 Unwinding the DNA Double Helix Requires DNA Helicases,
Topoisomerases, and Single-Stranded DNA Binding Proteins
• During DNA replication the two strands of the double helix must unwind at each
replication fork. Three classes of proteins facilitate the unwinding: DNA helicases,
topoisomerases, and single-stranded DNA-binding proteins (SSB).

• DNA helicases are responsible for unwinding the DNA, using energy from ATP
hydrolysis. The unwinding of the helix would create too much supercoiling and
possibly tangling the rest of the DNA if not for the actions of topoisomerases.

• SSB attach to the exposed single strands to keep the DNA unwound and therefore
accessible to the DNA replication machinery.

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Mitosis Is Subdivided into Prophase, Prometaphase,
Metaphase, Anaphase, and Telophase
• Mitosis is divided into five stages based on changing appearance and behavior
of chromosomes

• Events during each stage are directed toward the correct distribution of one
copy of each chromosome into daughter nuclei

Chromosomes in mitosis
• At the beginning of mitosis, chromatin folds and condenses to produce visible
chromosomes

• DNA has already replicated, so each chromosome is composed of two sister

chromatids

• The microtubules of the mitotic spindle will distribute the chromatids to

opposite ends of the cell

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Prophase

• After DNA replication, cells exit S phase and enter G2 phase, where final
preparations are made for entry into mitosis

• Toward the end of G2, chromosomes begin to condense into more compact,
folded structures

• The G2 → prophase transition is not sharply defined but cells are in prophase
when individual chromosomes become visible

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 Prophase
• In prophase, each chromosome
has two chromatids

• The centrosomes near the

nucleus, which function as
microtubule organizing centers
(MTOC), begin to migrate away
from each other.
• A dense starburst of MTs called
an aster forms near each
centrosome
• Within the centrosomes are • As centrosomes move, they act as nucleation
microtubule-containing sites for microtubules (MTs). The microtubule-
structures called centrioles containing apparatus is responsible for
separation of chromatids into daughter cells

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 Prometaphase

• The onset of prometaphase is marked by the fragmentation of the

membranes of the nuclear envelope

Spindle assembly
• During late prophase, MT growth speeds up dramatically and initiation of
new MTs at centrosomes increases

• When the nuclear envelope disintegrates, kinetochores and MTs can come
into contact

• When the plus end of MTs and the kinetochore bind, the MT becomes a
kinetochore MT

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 Centrosomes complete their movement to
opposite sides of the nucleus and the spindle
MTs contact the condensed chromosomes

Spindle MTs attach to paired chromatids in the

kinetochore

• Kinetochore is formed by the assembly of

proteins at the centromere.
• Kinetochore begin to form shortly after S phase.

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 Chromosome Alignment and Separation
• Chromosomes migrate to the central region of the spindle through a series of
movements generated by pulling and pushing forces from the kinetochore
microtubules - Congression

• Chromosomes reach the central region and stay there as a result of precisely
balanced forces pulling them toward opposite poles

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 Metaphase
• A cell is in metaphase when the fully condensed
chromosomes are all aligned at the metaphase plate
(a plane equidistant between the two poles of the
spindle)

• Examining metaphase cells allows chromosomes to

be identified, generating a karyotype

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 Spindle Assembly and Chromosome Attachment
• Microtubules have an inherent polarity (the two ends have
different chemical properties)

• The end where MT assembly is initiated (the centrosome in the

case of the spindle) is the minus end

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 Anaphase
• Anaphase is the shortest phase of mitosis

• The two sister chromatids of each

chromosome abruptly separate and move
toward opposite poles

• the chromosomes are pulled toward

spindle poles as kinetochore MTs get
shorter

• The 2 spindle poles move away from

each other as polar MTs lengthen

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 Roles of motor proteins in chromosome movement

• 1. Chromosomes are moved,

kinetochores first, toward the spindle
poles during anaphase

• This is driven by kinesins associated

with the kinetochore MTs

• One kinesin-like motor is at the plus end

of the MT, embedded in the kinetochore,
and moves the chromosome as it
“chews up” the MT

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• 2. Motor proteins play a role in the
movement of the spindle poles away from
each other

• Bipolar kinesin motors bind to

overlapping polar MTs from opposite
spindle poles, forcing the spindle poles
away from each other

• 3. Cytoplasmic dynein is associated with

astral microtubules that are connected to
the cell cortex

• The cell cortex is a layer of actin

microfilaments lining the inner surface of
the plasma membrane

• The dynein moves toward the minus

ends of the microtubules and appears to
move the spindle toward© 2012
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 Motor Proteins and Chromosome Movement

• Several motor proteins play active roles in mitosis

• They use energy from ATP to change shape and exert force that
causes movement of attached structures

• Motor proteins play at least three distinct roles in movement of

anaphase chromosomes

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 Telophase

• At the beginning of telophase the daughter

chromosomes arrive at the poles of the spindle

• Chromosomes uncoil into interphase chromatin

• Nucleoli reappear and nuclear envelopes reform

• During this period, cytokinesis also takes place

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Cytokinesis Divides the Cytoplasm

• After the chromosomes have separated,

cytokinesis divides the cytoplasm in two

• It starts in early telophase

• Cytoplasmic division is called
cleavage; it begins with a slight
puckering of the cell surface that
deepens into a cleavage furrow

• The furrow continues to deepen until

opposite surfaces make contact and
split the cell in two

• The furrow deepens along a plane

passing through the spindle equator

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 Regulation of the Cell Cycle
• Variations are observed in
– overall length of the cell cycle
– relative length of time spent in various phases
– how closely mitosis and cytokinesis are coupled

• The cell cycle is regulated to meet the needs of each cell type and organism

Length of the Cell Cycle Varies Among Different Cell Types

• At one extreme are cells that divide continuously to replace cells that are
constantly lost or destroyed
– e.g., cells involved in sperm formation, and stem cells that give rise to
blood cells, skin cells, and epithelial cells that from the inner lining of
lungs and intestines

• At the other extreme are extremely slow growing tissues or even some
(mature nerve or muscle cells) that do not divide at all
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Variations in generation time

• Most of the variations in generation time are based on differences in the

length of G1, though S and G2 can also vary

• Cells that divide very slowly may spend days, months, or years in the
offshoot of G1 called G0

• Cells that divide very rapidly have a short G1 or may skip it entirely

Cell growth and the cell cycle

• Cell growth and cell cycle are linked in general, although there are exceptions.

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Progression Through the Cell Cycle Is Controlled at
Several Key Transition Points/ Checkpoint
• Control of the cell cycle must

– 1. Ensure that events of each phase are carried out in the correct order
and at the appropriate time

– 2. Ensure that each phase is completed before the next one begins

– 3. Respond to external conditions

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 Key transition points/ Checkpoints
• At key transition points in the cell cycle,
conditions in the cell determine whether or
not it will proceed

• The first control point occurs in late G1; the

G1→ S progression.

• In animal cells, the comparable control

point is called the restriction point; the
ability to pass it is influenced by the
presence of extracellular growth factors

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 First Key transition points
• Cells that successfully pass the
restriction point are committed to S
phase

• Those that do not, enter into G0 and

reside there until a signal allows them to
reenter G1 and pass through the
restriction points

• The DNA replication checkpoint

ensures that DNA synthesis is complete
before the cell exits G2 and begins
mitosis
The DNA damage checkpoint
• A multiple series of DNA damage checkpoints
monitor DNA for damage and halt the cell
cycle at various points (late G1, S, and late G2)
by inhibiting different Cdk-cyclin complexes
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second transition point

• Another transition point occurs at the

G2 à M boundary, where the
commitment is made to enter mitosis

• In some cell types, the cell can be

arrested in G2 indefinitely and the cell
enters a state analogous to G0

• In most cells the G1 arrest is the more

prevalent type of control

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third transition point

• A third important transition point is during

M phase, between metaphase and
anaphase

• Here the commitment is made to move

the two sets of chromosomes into the
new cells

• Before cells can begin anaphase, all the

chromosomes must be properly attached
to the spindle

• The mitotic spindle checkpoint

prevents anaphase from beginning before
the chromosomes are all attached to the
spindle

• Kinetochores that remain unattached to

microtubules produce a “wait” signal
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 DNA repair Mechanism :
1) Errors introduced by DNA polymerase
Corrected by the intrinsic 3’-5’ exonuclease activity of DNA polymerase itself.
If error escape proofreading – mistmatch repair will be initiated.

2) Damaged bases encountered during DNA replication (S phase):

Translesion synethesis: Specific DNA polymerase allows synthesis of
new DNA across regions in which the DNA template is damaged – A
damage-tolerated mechanism that prevent an initial mutation from being
passed on to newly forming DNA strand.

3) Damage found after DNA replication

- base excision repair: correct single damaged base in DNA.
- nucleotide excision repair: correct bulky lesion.
- Mismatch repair: correct noncomplementary base pairs

4) Double-Strand DNA Breaks

Nonhomologous End-Joining – can be used any time.
Homologous Recombination – Only used during DNA replication (S phase)

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1) Proofreading Is Performed by the 3¢→ 5¢ Exonuclease
Activity of DNA Polymerase
• About 1 of every 100,000 nucleotides incorporated during DNA replication is
incorrect. Therefore, there will be ~ 120,000 errors every time a human cell
duplicates its DNA. Such mistakes are usually fixed by a proofreading
mechanism.

• Almost all DNA polymerases have a 3¢ → 5¢ exonuclease activity

• Exonucleases degrade nucleic acids from the ends of the molecules. The
exonuclease activity of DNA polymerase allows it to remove incorrectly base-
paired nucleotides and incorporate the correct base

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 Environmental hazards lead to DNA damage

• DNA must be accurately passed on to daughter cells

• In addition to ensuring that replication is faithful, this also means that DNA
alterations must be repaired

• DNA alterations, or mutations, can arise spontaneously, or through exposure

to environmental agents

DNA damage by mutagens

• DNA damage can be caused by mutation-causing agents, mutagens

• Environmental mutagens fall into two categories: chemicals and radiation

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 DNA Damages that Occur Spontaneously

• During DNA replication, some types of

mutations occur through spontaneous
hydrolysis reactions:

• Depurination is loss of a purine base (A

or G)

• Deamination is removal of a base’s

amino group, changing its base-pairing
properties

• Deamination may involve A, G, or, most

often, C

• If not corrected, Uracil will behave like at

T in the next round of DNA replication.

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 DNA damage induced by mutation causing agents:
1) Chemicals, and 2) Radiation.

Chemicals:
• Base analogs: resemble nitrogenous bases and are incorporated into DNA.
Eg, 5-bromouracil

• Base-modifying agents: react chemically with DNA bases to alter their

structures, forming DNA adducts.
• Intercalating agents: insert themselves between adjacent bases, distorting
DNA structure.

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 Radiation:

• Ultraviolet radiation alters DNA by

triggering pyrimidine dimer formation –
covalent bonds between adjacent
pyrimidine bases

• X-rays radiation, called ionizing

radiation, remove electrons from
molecules, and generate highly reactive
intermediates that damage DNA
(induces DNA breaks)

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 Mechanisms that repair damaged DNA:

2) Translesion Synthesis Correct Mutations Involving Abnormal

Nucleotides

Translesion Synthesis:

Use to repair pyrimidine dimers and abasic (deaminated or

depurinated base).

Is NOT a mechanism to repair DNA damage. Instead, it is a way to

ignore the damage, allowing replication to take place.

• Repair takes place during replication, using translesion DNA

polymerases. Able to put the right nucleotides in the strand
complementary to the damaged strand

•Repair damage in the new strand but not in the template strand –
prevent the mutation being passed onto newly formed DNA strand.

http://www.youtube.com/watch?v=gDc6pAH72w0

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3) Excision Repair Correct Mutations Involving Abnormal Nucleotides
Excision Repair pathway:

•Errors remaining after DNA replication are repaired

by excision repair, in which abnormal nucleotides are
removed and replaced.

•Players: DNA damage recognition proteins/Repair

endonuclease/DNA polymerase/DNA ligase.

•Three-step process:

Repair endonucleases are recruited to DNA by

proteins that recognize damage

1. They cleave the backbone adjacent to the damage

site; other enzymes remove the defective nucleotides

2. DNA polymerase replaces the missing nucleotides,

using the complementary strand as template

3. DNA ligase seals the remaining nick in the repaired

strand

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 Types of excision repair

• Excision repair pathways are classified into two types:

• A) Base excision repair: corrects single damaged bases

• E.g., deaminated bases are detected by DNA glycosylases, which

recognize and remove the base by cleaving the bond between the
base and the sugar

• The sugar with the missing base is then recognized by a repair

endonuclease that detects depurination

• It breaks the phosphodiester backbone to one side of the sugar and a

second enzyme removes the sugar

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 B) Nucleotide excision repair (NER)
• For removing pyrimidine dimers and other bulky lesions, a second type
of excision repair is employed

• Nucleotide excision repair uses proteins that detect distortions in the

DNA helix and recruit NER endonuclease (or exinuclease) that cuts the
DNA backbone on either side of the lesion

• Helicase unwinds the DNA between the nicks, and frees it from the
DNA; DNA polymerase and ligase complete the repair.

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C) Mismatch Repair Corrects Mutations That Involve
Noncomplementary Base Pairs
Noncomplementary Base Pairs: eg, A:G, T:C,…..

• Mismatch repair targets errors made during DNA replication that

escape proofreading.

• No structural variation in noncompletmentary base pairs.

• Mismatch repair is able to distinguish the original vs. the newly

synthesized strand in order to correctly repair the mismatch. Otherwise
would create a permanent mutation.

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 Double-Strand DNA Breaks Are Repaired by Nonhomologous End-
Joining or Homologous Recombination

• Double-strand breaks cleave DNA into two fragments

• It is difficult for the repair system to identify and rejoin the correct
broken ends without loss of nucleotides

• Two pathways are used: nonhomologous end-joining and

homologous recombination

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MRN:
Mre11, Rad50, Nbs1

Homologous recombination Nonhomologous end-joining

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 Nonhomologous end-joining (NHEJ)
• Direct ligation of broken ends without the need for a homologous template.

• Nonhomologous end-joining uses a set of proteins that bind to ends of

broken DNA fragments and join them together

• This is error-prone, because nucleotides can be lost from the broken ends,
and there is no way to ensure the correct DNA fragments are joined.

• NHEJ is predominant in the G1 phase of the cell cycle, when the cell is
growing but not yet ready to divide.
Homologous recombination
• Homologous recombination is a more precise method for fixing double-
strand breaks

• If the DNA molecule from one chromosome is broken, the homologue is

available as a template to guide accurate repair.

• Repairs DNA before the cell enters M phase. It occurs during and
shortly after DNA replication, in the S and G2 phases of the cell cycle,
when sister chromatids are more easily available.
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 Putting It All Together: The Cell Cycle Regulation
Machine
• The “machine” that regulates the
eukaryotic cell cycle involves two
interacting mechanisms
– 1. An autonomous clock goes
through a fixed cycle over and
over again via the synthesis and
degradation of cyclins
– 2. The clock is adjusted as
needed
Cell cycle progression is driven
by protein kinases that are
active only when bound to an
activator protein called cyclin

These kinases are cyclin-

dependent kinases (Cdks)

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