This was a journal club presentation I made. I don't remember what it was specifically about.

1. DNA and Stuff…
By:

2. Meanings of words in the Title in the
context of said title
The - definite article
Mammalian - pertaining to the class Mammalia; characteristic of mammals
DNA - deoxyribonucleic acid: an extremely long macromolecule that is the main component of
chromosomes and is the material that transfers genetic characteristics in all life forms
Replication - the process by which double-stranded DNA makes copies of itself
Elongation – the process during transcription by which RNA is made from a compliment DNA
strand
Checkpoint - a place along a road, border, etc., where travelers are stopped for inspection
Implication - something implied or suggested as naturally to be inferred or understood
of
Chk1 - CHK1 is one of two serine/threonine kinases involved in the induction of cell cycle
checkpoints in response to DNA damage and replicative stress.
and - conjunction
Relationship - a connection, association, or involvement.
with - accompanied by; accompanying
Origin Firing - commencement at the beginning of
As - in the manner
Determined by Single DNA Molecule and Single Cell Analyses – what we do

3. But Ant… I don’t understand all that
fancy talk you just said.

4. Let’s Start with Elongation
According to some, this is the step of
transcription (the whole process of
initiation, elongation, and termination
of RNA synthesis) where a template
strand of DNA is used to encode RNA in
RNA synthesis.
An RNA Polymerase (the globule that
reads the DNA strand and adds
nucleotides to the RNA strand) reads a
template from the 3’-> 5’ end but is
said to transcribe 5’-> 3’ because it is a
duplicate of the coding strand (the non-
template strand).
This means biologists make things
Regulation of Elongation (fork velocity)
annoying for us even though they think
it makes it simpler. Why is it called this? Because those pictures
above look like forks.

5. Now on to CHK1
CHK1 and CHK2 are two serine/threonine kinases involved in the induction of cell cycle
checkpoints in response to DNA damage and replicative stress. CHK1 is essential for the G2/M
DNA damage-induced checkpoint, and inhibitors of this kinase are expected to lead to
sensitisation of tumours to both radiotherapy in the form of ionising radiation (IR) and
chemotherapeutic agents which are used in the clinic for the treatment of cancer. Recent
studies suggest that unlike CHK1, CHK2 is not essential for the initiation of the G2
checkpoint, and there is evidence of a functional link between the tumour suppressor p53 and
CHK2. In this context, CHK2 inhibition would be predicted to reduce p53-induced apoptosis and
protect normal tissues from chemotherapy. The recent discovery that activated CHK2
phosphorylates the pro-apoptotic transcription factor E2F resulting in protein
stabilisation, activation and E2F-dependent apoptosis may still provide a therapeutic strategy for
increasing tumour cell susceptibility to DNA damage. Inhibition of CHK2 in cancer cells with
defective checkpoints, may allow tumour cells to remain proliferating and thus become more
sensitive to IR or chemotherapy. Assays have therefore been developed in order to screen for
inhibitors of both CHK1 and CHK2 and a number of hits have been identified that are now
undergoing evaluation for further drug development. A collaboration has also been initiated
with Professor L Pearl (Section of Structural Biology) to crystallise the CHK2 kinase, to aid drug
development on this project.
That’s a mouth full!.?,;:*

6. Simply Stated…
A kinase -
a type of enzyme that transfers phosphate
groups from high-energy donor
molecules, such as ATP, to specific target
molecules
CHK1
G2/M checkpoints include the checks
for damaged DNA, unreplicated
DNA, and checks that ensure that the
genome is replicated once and only
once per cell cycle. If cells pass these
checkpoints, they follow normal
Used in G2/M What is transition to the M phase. However, if
checkpoint. this? any of these checkpoints fail, mitotic
entry is prevented by specific G2/M
checkpoint events

7. Not my work
CELL CYCLE CHECKPOINTS
CYCLIN E / cdk2
CYCLIN A/ cdk2
S
G1 G2
CYCLIN D / cdk4,5,6
M
CYCLIN B/ cdc2

8. Not my work
CELL CYCLE CHECKPOINTS
• MITOSIS ENTRY (G2/M)
– Replication Complete
– Growth/ Protein Synthesis adequate
– No DNA Damage
• S-PHASE ENTRY (G1/S)
– Mitosis Complete ?signal - cyclin
degradation
– Growth/ Protein Synthesis (G1 CYCLINS)
– No DNA Damage
• OTHERS
– MITOSIS EXIT: ?coupling to S-phase
– S PHASE : coupling to mitosis
• also in response to DNA damage
– G1 sequence of events
• signaling from cell surface

9. Not my work
DNA Damage - Cell Cycle Arrest
damage dependent checkpoints
G1 - S - G2 G1 - S - G2
CELL
wild-type
No.
DNA content DNA content
X-ray treated
asynchronous G1/S block loss of G1/S in
p53 deficient
G2/M block cells
(6-9 hours)

10. Not my work
G2/M CHECKPOINT IN
RESPONSE TO DAMAGE
• MAMMALIAN CELLS
– chk1/chk2 kinases involved
– ?upstream pathway uncertain ?ATM ?ATR
– many other proteins: e.g. 9-1-1 complex
• YEAST
– RAD9: radiation sensitive (resistant with MBC = G2
hold)
• cf RAD52 = radiation sensitive +/- MBC
– RAD17,24; MEC3 = late S signal (RAD9)
– MEC1,2 = mitosis entry (late S or pre-S)

11. Not my work
S PHASE CHECKPOINT IN
RESPONSE TO DAMAGE
• MAMMALIAN CELLS
– involves Rad50/mre11/NBS1
– ?upstream pathway uncertain ?ATM ?ATR
– many other proteins: ?BRCA1?; 9-1-1 complex
• YEAST (pombe)
– RAD9/ RAD1/ Hus1:
– cdc18
– Rad17, Rad24

12. Is your hair a mess?
Is it hard to comb?
Well do we have the solution for you…
What a
mess!!
It’s
new!!

14. Welcome Back
• Time for some pictures…

15. Figure 2
Figure 2. The inter-origin distance is linearly
correlated with the velocity of neighboring
forks. (A and B) Schemes representing two
DNA fibers with three and two origins of
replication. The forks travel faster when only
two origins are activated, depicted by longer
green and red signals, because a longer
segment of DNA must be duplicated in the
same amount of time. (C) Linear correlation
between inter-origin distance and fork velocity
for neighboring origins. The dot plot, which
supports the scheme shown above, is obtained
by the analysis of primary keratinocytes, where
a linear correlation of 0.65 was calculated.19
(D and E) Model depicting two replication foci
where each DNA fiber is folded into a
replication factory. The left factory (D) contains
smaller loops, with denser origins and slower
forks than the right factory (E).10

16. Figure 3

17. Figure 4
Figure 4. Single-cell analyses of the DNA replication
checkpoints. (A) Confocal microscopy images of
representative cells immediately after CPT treatment
(HT29 cells were incubated with IdU for 15 minutes, then
1 mM CPT was added for an additional 30 minutes). The
top panels depict IdU-labeled replication foci patterns in
early, mid, and late S phase. The bottom panels depict
g-H2AX foci colocalizing with replication foci. (B)
Sequential pulse-labeling of replication foci for
untreated, CPT-treated, and CPT+CHIR124-treated cells.
Representative cells at different times are shown. In
untreated conditions, when a time lapse is introduced
between the two pulses, the red and green foci are
separate entities, indicating that new origins (in red) were
activated at new sites during the second pulse, while the
foci labeled during the first pulse have completed
replication (no IdU incorporation into these pre-existing
sites). After CPT treatment, the second pulse is not
incorporated, indicating that new origins are inhibited.
Administration of the Chk1 inhibitor CHIR124 (0.1 mM)
restored the incorporation of IdU and origin firing.10