1) DNA repair mechanisms are responsible for minimizing damage to DNA from various sources. When DNA damage surpasses a threshold, cells can enter senescence, apoptosis, or become cancerous. 2) There are several DNA repair mechanisms, including base excision repair, nucleotide excision repair, and mismatch repair for single-strand damage, as well as nonhomologous and homologous end joining for double-strand breaks. 3) The cell cycle is regulated by checkpoints at the G1/S and G2/M transitions, which are controlled by complexes of cyclin-dependent kinases (CDKs) and cyclins. CDK-cyclin complexes phosphorylate proteins to drive the cell cycle forward past

1. DNA Repair
Mechanisms &Cell
CycleCheckpoints
- Surendranath Reddy (International European University)
Surendranath Reddy

2. Do you remember the DNA
structure?
Surendranath Reddy

3. What is DNA repair?
 A set of processes for repairing the many
accidental lesions that occur continually in
DNA
 Lesion is a region in an organ or tissue which
has suffered damage through injury or
disease, such as a wound, ulcer, abscess, or
tumour.
 What if your DNA is damaged? How can it be
damaged?
Surendranath Reddy

4. DNA Repair (Continued..)
 DNA repair mechanisms are responsible for minimizing the negative
effects that DNA damage has on the cell. DNA damage occurs almost
constantly in living cells.
 When DNA damage surpasses a certain threshold, either because there is
too much accumulated damage or because DNA repair mechanisms are
no longer effective, a cell can have one of the following three fates:
1. Senescence: A cell enters a dormant state that is irreversible, in
which the main cellular processes and functions are suspended.
2. Apoptosis: A cell undergoes programmed cell death, or suicide, by
activating specialized signal cascades.
3. Cancer: A cell starts undergoing unregulated cell division, resulting
in neoplasia and tumour growth.
DNA repair is thus extremely important for proper functioning of cells
and the organism as a whole. A number of specialized DNA repair
mechanisms have evolved, lets discuss some.
Surendranath Reddy

5. Types of DNA
Repair Mechanisms
Surendranath Reddy

6. This is how DNA is getting
damaged
Surendranath Reddy

7. SingleStrand
Damage
 BASE EXCISION REPAIR
When single nucleotides are
damaged by alkylation,
deamination, or oxidation
reactions, two enzymes, DNA
glycosylase and AP
endonuclease remove and repair
the damaged bases.
Endonuclease nicks the
phosphodiester bond next to the
base, releases deoxyribose, and
creates a gap.
DNA polymerase then inserts
the correct nucleotide in its place
(based on the complementary
base), and the nick is sealed by
DNA ligase.
The most common DNA damage
is the deamination of cytosine to
uracil.
Surendranath Reddy

8. SingleStrand
Damage
 NUCLEOTIDE EXCISION
REPAIR
A set of mechanisms similar to
base excision repair, but used
to excise and replace longer
stretches of nucleotides (2–30
bases).
UV-damaged DNA: dimers
form between adjacent
pyrimidines (e.g., thymine),
thus preventing DNA
replication.
UV-specific endonuclease
(uvrABC excinuclease)
recognizes the damaged base
and makes a break several
bases upstream (toward the 5′
side).
Surendranath Reddy

9. SingleStrand
Damage
 MISMATCH REPAIR
Mismatch repair is used when
there is an error in the pairing
of nucleotides secondary to
DNA replication or
recombination.
The base pair mismatch repair
system detects errors that
escaped proofreading during
DNA replication
Surendranath Reddy

10. DoubleStrand
Damage
 NONHOMOLOGOUS END
JOINING
When both strands of DNA are
broken in a region that has not
yet been replicated, there is truly
no template for the cell to use to
reconstruct the damaged DNA.
However, because a complete
break of the DNA double helix is
highly deleterious for the cell, an
attempt is made to fix the break
using NHEJ.
In this process, DNA ligase–
containing complexes join the
separated ends of the double
helix, relying on
microhomologies between the
ends of the singlestranded
fragments. However, by defi
nition, NHEJ is always
mutagenic..
Surendranath Reddy

11. DoubleStrand
Damage
 HOMOLOGOUS END
JOINING
(RECOMBINATORIAL
REPAIR)
Sometimes a double-stranded
break occurs during DNA
replication.
In this case, a fragment of the
DNA has already been
replicated and can serve as a
template for the repair of the
double-stranded break.
Molecularly, the enzymatic
complex involved in
recombinatorial repair is
similar to that involved in
chromosomal crossover.
Surendranath Reddy

12. CellCycle
Checkpoints
Surendranath Reddy

13. Checkpoint control
system
 Checkpoints
 cell cycle controlled by STOP &
GO chemical signals at critical
points
 signals indicate if key cellular
processes have been completed
correctly
Surendranath Reddy

14. Checkpoint
control system
 3 major checkpoints:
 G1/S
 can DNA synthesis begin?
 G2/M
 has DNA synthesis been completed
correctly?
 commitment to mitosis
 spindle checkpoint
 are all chromosomes attached to spindle?
 can sister chromatids separate correctly?
Surendranath Reddy

15. G1/S
checkpoint
 G1/S checkpoint is most critical
 primary decision point
 “restriction point”
 if cell receives “GO” signal, it divides
 internal signals: cell growth (size), cell nutrition
 external signals: “growth factors”
 if cell does not receive
signal, it exits cycle &
switches to G0 phase
 non-dividing, working state
Surendranath Reddy

16. G0 phase
M
Mitosis
G1
Gap 1
G0
Resting
G2
Gap 2
S
Synthesis
 G0 phase
 non-dividing, differentiated state
 most human cells in G0 phase
 liver cells
 in G0, but can be “called back” to
cell cycle by external cues
 nerve & muscle cells
 highly specialized
 arrested in G0 & can never divide
Surendranath Reddy

17. Cdk / G1
cyclin
Cdk / G2
cyclin (MPF)
G2
S
G1
C
M
G2 / M checkpoint
G1 / S checkpoint
APC
Active
Inactive
Active
Inactive
Inactive
Active
mitosis
cytokinesis
MPF = Mitosis
Promoting Factor
APC = Anaphase
Promoting Complex
• Replication completed
• DNA integrity
Chromosomes attached at
metaphase plate
Spindle checkpoint
• Growth factors
• Nutritional state of cell
• Size of cell
Surendranath Reddy

18. The basic
mechanism is
quite simple.
Surendranath Reddy

19. The basic
mechanism is
quite simple.
Surendranath Reddy

20. Cyclins &Cdks
 Interaction of Cdk’s & different cyclins triggers the stages of
the cell cycle
Leland H. Hartwell
checkpoints
Tim Hunt
Cdks
Sir Paul Nurse
cyclins
1970s-’80s | 2001
Surendranath Reddy

21. Cyclin-
dependent
protein
kinases(Cdks)
 Cyclin-dependent protein kinases (Cdks) are enzymes that
phosphorylate (add phosphate groups to) the serine and threonine
amino acids of key cellular enzymes and other proteins.
 At the G2 checkpoint, for example, Cdks phosphorylate histones,
nuclear membrane filaments, and the microtubule-associated
proteins that form the mitotic spindle.
 Phosphorylation of these components of the cell division machinery
initiates activities that carry the cycle past the checkpoint into mitosis.
 Cyclins are proteins that bind to Cdks, enabling the Cdks to function as
enzymes. Cyclins are so named because they are destroyed and
resynthesized during each turn of the cell cycle. Different cyclins
regulate the G1 and G2 cell cycle checkpoints.
Surendranath Reddy

22. How cell cycle control works?
Surendranath Reddy

23.  The G2 Checkpoint:
 During G2, the cell gradually accumulates G2 cyclin (also called
mitotic cyclin).This cyclin binds to Cdk to form a complex called
MPF (mitosis-promoting factor).
 At first, MPF is not active in carrying the cycle past the G2
checkpoint. But eventually, other cellular enzymes phosphorylate
and so activate a few molecules of MPF.These activated MPFs in
turn increase the activity of the enzymes that phosphorylate MPF,
setting up a positive feedback that leads to a very rapid increase in
the cellular concentration of activated MPF.When the level of
activated MPF exceeds the threshold necessary to trigger mitosis,
G2 phase ends.
 MPF sows the seeds of its own destruction.The length of time
the cell spends in M phase is determined by the activity of MPF, for
one of its many functions is to activate proteins that destroy cyclin.
As mitosis proceeds to the end of metaphase, Cdk levels stay
relatively constant, but increasing amounts of G2 cyclin are
degraded, causing progressively less MPF to be available and so
initiating the events that end mitosis.After mitosis, the gradual
accumulation of new cyclin starts the next turn of the cell cycle
Surendranath Reddy

24.  The G1 Checkpoint:
 The G1 checkpoint is thought to be regulated in a
similar fashion. In unicellular eukaryotes such as
yeasts, the main factor triggering DNA replication
is cell size.
 Yeast cells grow and divide as rapidly as
possible, and they make the START decision by
comparing the volume of cytoplasm to the size of
the genome.
 As a cell grows, its cytoplasm increases in size,
while the amount of DNA remains constant.
Eventually a threshold ratio is reached that
promotes the production of cyclins and thus
triggers the next round of DNA replication and cell
division.
Surendranath Reddy

25. CellCycle
Regulation
 The cell cycle is regulated at the G1/S and G2/M boundaries
(checkpoints) by phosphorylation of complexes of a protein kinase
[cyclin-dependent kinase (Cdk) protein] and a cyclin (cytoplasmic
oscillator).
 For example, the G2/M interface is regulated by M-Cdk complex
(formerly called Mitosis Promoting Factor, MPF), which is
responsible for the phosphorylation of spindle proteins, histones,
and lamins.
 Phosphorylation of lamins results in their breakdown as well as the
dissolution of the nuclear envelope.There are different cyclins and
Cdks for each of the cell cycle checkpoints.
 Overarching the Cdks are the Cdk inhibitors that form an
additional regulatory layer at each of the cell cycle checkpoints.
Study of the cell cycle is critical to an understanding of the
regulation of abnormal proliferation as occurs in cancer cells.
Surendranath Reddy

26. Retinoblastoma
& p53
 Two tumour suppressor genes that have been well studied are
retinoblastoma gene (Rb) and p53.
 Rb is active (suppressing growth) in the hypo phosphorylated state and
inactive in the hyperphosphorylated form.
 In its non phosphorylated form Rb serves as a brake on the cell cycle at the
G1/S interface by binding to the transcription factor, E2F.
 Stimulation by growth factors results in phosphorylation and release of the
brake; E2F is free to turn on transcription of cell cycle genes, allowing cells
to traverse the G1/S interface.
 Mutations in Rb occur in tumours; a mutation has the same effect as
inactivating Rb leading to uncontrolled cell proliferation as E2F transcribes
cell cycle genes.
 p53 is a protective gene or molecular policeman, which prevents the
replication of damaged DNA and stimulates repair.
 p53 acts as a transcription factor and also works through the Cdk inhibitors
to arrest the cell cycle at the G1/S interface. p53 mutations are found in
many human tumours
Surendranath Reddy

27. References
 Radiobiology for the Radiologist 7th Edition
-by Eric J. Hall (Author), Amato J. Giaccia (Author)
 Radiation Biology of Medical Imaging
-Charles A. Kelsey PhD,, Philip H. Heintz PhD,, Daniel J.
Sandoval MS,, Gregory D. Chambers MS,, Natalie L. Adolphi
PhD,, Kimberly S. Paffett MS
 Cell Cycle Checkpoints (Methods and Protocols)
- Willis X. Li
 Molecular Biology of the Cell – Bruce Alberts
 Other References such as books and GOOGLE,..
Surendranath Reddy

28. ThankYou
Surendranath Reddy