DNA and Stuff…

By:

The - definite articleMammalian - pertaining to the class Mammalia; characteristic of mammalsDNA - deoxyribonucleic acid: an extremely long macromolecule that is the main component of chromosomes and is the material that transfers genetic characteristics in all life formsReplication - the process by which double-stranded DNA makes copies of itselfElongation – the process during transcription by which RNA is made from a compliment DNA strandCheckpoint - a place along a road, border, etc., where travelers are stopped for inspectionImplication - something implied or suggested as naturally to be inferred or understoodof 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 - conjunctionRelationship - a connection, association, or involvement.with - accompanied by; accompanyingOrigin Firing - commencement at the beginning ofAs - in the mannerDetermined by Single DNA Molecule and Single Cell Analyses – what we do

Meanings of words in the Title in the context of said title

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

Let’s Start with ElongationAccording 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 annoying for us even though they think it makes it simpler.

Regulation of Elongation (fork velocity)Why is it called this? Because those pictures above look like forks.

Now on to CHK1CHK1 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!.?,;:*

Simply Stated…a type of enzyme that transfers phosphategroups from high-energy donor molecules, such as ATP, to specific target molecules

A kinase -

CHK1

Used in G2/M checkpoint.

What is this?

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

transition to the M phase. However, if any of these checkpoints fail, mitotic entry is prevented by specific G2/M

checkpoint events

CELL CYCLE CHECKPOINTS

G1

S

G2

MCYCLIN B/ cdc2

CYCLIN A/ cdk2

CYCLIN E / cdk2

CYCLIN D / cdk4,5,6

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

Not my work

DNA Damage - Cell Cycle Arrestdamage dependent checkpoints

CELLNo.

DNA content DNA content

asynchronousX-ray treatedG1/S blockG2/M block(6-9 hours)

G1 - S - G2

wild-type

loss of G1/S in p53 deficientcells

G1 - S - G2

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)

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

Not my work

Is your hair a mess? Is it hard to comb?

Well do we have the solution for you…

What a mess!!

It’s new!!

Welcome Back

• Time for some pictures…

Figure 2Figure 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

Figure 3

Figure 4Figure 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 thefoci 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